Gene Editing Of Human IPSCs Rescues Thrombophilia In Hereditary Antithrombin Deficiency In Mice

Objective:Hereditary antithrombin deficiency is a congenital coagulation disorder caused by SERPINC1 gene mutations and predisposes to recurrent venous thromboembolism that can be life-threatening.Therefore,lifelong anticoagulation is required,which has side-effects and may not always be effective.The objective of this study was to explore the possibility of gene editing and cellular therapy mediated by induced pluripotent stem cells（iPSCs）to improve the propensity for thrombosis in hereditary AT deficiency.Methods:A patient with severe hereditary AT deficiency who carries a homozygous point mutation in SERPINC1 gene was recruited for this study.Peripheral blood mononuclear cells were extracted from the patient and reprogrammed into iPSCs by Sendai virus transfection.CRISPR-Cas9 and Cre/Lox P genome editing tools were used to correct the mutation of the iPSCs,and then the normal iPSCs after gene editing were differentiated into mature hepatocytes.Finally,the hepatocytes were transplanted into antithrombin-knockout mice by spleen injection.Changes in plasma AT activity and concentration were monitored after transplantation,as well as to assess whether the thrombophilia of AT-deficient mice was improved.Results:After transplantation of i PSC-differentiated hepatocytes,the activity and concentration of plasma AT in AT^+/-KO mice remained high for 3 weeks.Compared with untreated AT^+/-KO mice,the plasma AT expression of AT^+/-KO mice returned to normal level at the second week after hepatocytes transplantation（activity:from 46.8±5.7%to 88.6±7.6%,p<0.0001;antigen:from 146.9±19.5 ng/m L to 390.7±16.1 ng/m L,p<0.0001）.In the inferior vena cava thrombosis model,the rate of venous thrombosis in mice treated with iPSCs-edited-Heps,iPSCs-parental-Heps,placebo and wild-type mice were 60%,90%,90%and 70%,respectively.The thrombus weight was much lighter（15.32±2.87 mg vs 7.25±2.00 mg,p=0.0025）and the thrombus length was much shorter（5.54±0.82 mm vs 2.70±1.24 mm,p=0.0015）in mice treated with iPSCs-edited-Heps compared with iPSCs-parental-Heps,and showed no significant difference compared with that in wild-type mice（weight:10.41±2.91 mg vs 7.25±2.00 mg,p>0.99;length:3.54±0.49 mm vs 2.70±1.24 mm,p>0.99）.The activity and concentration of antithrombin remained high for 3 weeks after injection.During the observation period of one month,no abnormal phenotype or death was observed in mice that after hepatocyte transplantation,and no obvious abnormal changes in liver and kidney function markers were found.Conclusion:The gene-corrected human iPSCs differentiated hepatocytes could successfully colonize the AT^+/-KO mice,stably and continuously secreted normal AT protein,which improved the blood hypercoagulation state and rescue thrombophilia in AT-deficient mice.This study confirms that the correction of mutations in patient-derived iPSCs and its mediated gene therapy are safe and effective,which provides potential curative applications for hereditary thrombophilia.