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

Abstract

ABSTRACTProtein C (PC) is a plasma anticoagulant encoded byPROC; mutation in bothPROCalleles 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 Sin 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 formationin vivoin C57BL/6 mice. The insertion ofEGFPsequence conjugated with self-cleaving peptide sequence atAlblocus via neonatalin vivogenome 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 editingin 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 SummaryEctopic expression of an engineered protein C via genome editing cures protein C deficiency in mice.