Cure of congenital purpura fulminans via expression of engineered protein C through neonatal genome editing in mice

Protein C (PC) is a plasma anticoagulant encoded by PROC ; mutation in both PROC alleles results in neonatal purpura fulminans—a fatal systemic thrombotic disorder. In the present study, we aimed to develop a genome editing treatment to cure congenital PC deficiency. First, we generated an engineered activated PC to insert a self-cleaving peptide sequence between light and heavy chains. The engineered PC could be released in its activated form and significantly prolonged the plasma coagulation time independent of the cofactor activity of protein S in vitro . The adeno-associated virus (AAV) vector-mediated expression of the engineered PC, but not wild-type PC, prolonged coagulation time owing to the inhibition of activated coagulation factor V in a dose-dependent manner and abolished pathological thrombus formation in vivo in C57BL/6 mice. The insertion of EGFP sequence conjugated with self-cleaving peptide sequence at Alb locus via neonatal in vivo genome editing using AAV vector resulted in the expression of EGFP in 7% of liver cells, mainly via homology-directed repair, in mice. Finally, we succeeded in improving the survival of PC-deficient mice by expressing the engineered PC via neonatal genome editing in vivo . These results suggest that the expression of the engineered PC via neonatal genome editing is a potential cure for severe congenital PC deficiency. One Sentence Summary Ectopic expression of an engineered protein C via genome editing cures protein C deficiency in mice. ### Competing Interest Statement TT, NB, YK, MH, NK, TH, and TO are inventors of the patent for the engineered PC sequence used in the present study. Other authors have no competing interests.