バータルツォグト ネメフバヤル

The repair of pathological gene variants is an ultimate aim for treating genetic diseases; however, the development of different therapeutic reagents for each of the many variants that can occur in a gene may not be scalable. Here, we investigated whether base editing to introduce a gain-of-function variant in blood coagulation factor IX (FIX) can increase FIX activity as a targeted therapeutic approach for hemophilia B. We engineered a G:C to A:T substitution at c.1151 of F9 by cytosine base editing to generate R338Q, known as the Shanghai F9 variant, which markedly potentiates coagulation factor activity. An adeno-associated virus vector harboring the base editor converted more than 60% of the target G:C to A:T and increased FIX activity in HEK293 cells harboring patient-derived F9 variants, as well as in knock-in mice harboring a human F9 cDNA. Furthermore, administration of lipid nanoparticles embedded with the base editor mRNA and gRNA increased FIX activity in mice. These data indicate that cytosine base editing to generate R338Q in FIX is a broadly applicable genome editing approach for hemophilia B with residual FIX activity.

BACKGROUND: Some patients with nonsevere hemophilia A (HA) are diagnosed with reduced factor (F)VIII activity (FVIII:C) relative to FVIII antigen (FVIII:Ag) and are classified as cross-reactive material-positive. We previously found a novel FVIII-Lys1693Asn (K1693N) natural mutation in a patient with HA with moderate phenotype (FVIII:C/FVIII:Ag = 1.9 IU/dL/124 IU/dL). OBJECTIVE: To examine the mechanism(s) involving the P4' site on thrombin cleavage site at Arg1689 in a cross-reactive material-positive phenotype. METHODS: FVIII-K1693N and its comparison variant K1693A were analyzed for t thrombin-mediated activation. FVIII-K376N, listed in the HA database due to a similar Lys-to-Asn substitution, was examined with K376A as its comparison. All mutants were expressed in BHK cells. RESULTS: FVIII-K1693N showed delayed cleavage at Arg1689 and low peak FVIII:C, while FVIII-K1693A showed mildly reduced FVIII:C after thrombin activation. Mildly reduced peak FVIII:C and similar cleavage to wild-type was illustrated, however, with the FVIII-K1693A mutation after thrombin activation. These findings indicated that the conversion to Asn at P4' site mediated thrombin resistance. FVIII specific activities of FVIII-K376N and FVIII-K376A were 6.1%/3.1% (one-stage assay) of wild-type, respectively. Thrombin-catalyzed cleavage at Arg372 was comparable to wild-type with both mutants, but thrombin-catalyzed FVIII activation was severely-depressed. Spontaneous decay rates of FVIIIa activity with FVIII-K376N (k = 0.53) and FVIII-K376A (k = 0.99) were greater compared to wild-type (k = 0.32), suggesting that cleavage of P4' site following cleavage at Arg372contributed to A1-A2 domainal interaction. CONCLUSION: Lys1693 and Lys376, both located at P4' sites, are suggested to influence FVIII function via distinct mechanisms, indicating that patient-derived mutations can reveal functional aspects of FVIII activation.

BACKGROUND: PC (protein C) 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. METHODS: We generated an engineered APC (activated PC) to insert a furin-cleaving peptide sequence between light and heavy chains. The engineered PC was expressed in the liver of mice using an adeno-associated virus vector or CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9)-mediated genome editing using an adeno-associated virus vector in vivo. RESULTS: The engineered PC could be released in its activated form and significantly prolonged the plasma coagulation time independent of the cofactor activity of PS (protein S) in vitro. The adeno-associated virus vector-mediated expression of the engineered PC, but not wild-type PC, prolonged coagulation time owing to the inhibition of activated coagulation FV (factor V) in a dose-dependent manner and abolished pathological thrombus formation in vivo in C57BL/6J mice. The insertion of EGFP (enhanced green fluorescent protein) sequence conjugated with self-cleaving peptide sequence at Alb locus via neonatal in vivo genome editing using adeno-associated virus 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. CONCLUSIONS: These results suggest that the expression of engineered PC via neonatal genome editing is a potential cure for severe congenital PC deficiency.