RNAi targeting LMAN1-MCFD2 complex promotes anticoagulation in mice.

Abstract

Combined deficiency of coagulation factor V (FV) and factor VIII (FVIII) is a rare bleeding disease caused by variants in either lectin mannose binding 1 (LMAN1) or multiple coagulation factor deficiency 2 (MCFD2) gene. Reducing the level of FVIII by inhibiting the LMAN1-MCFD2 complex may become a new anticoagulant approach. We aimed to find a new therapeutic option for anticoagulation by RNA interference (RNAi) targeting LMAN1 and MCFD2. siRNA sequences with cross-homology between mice and humans were designed based on LMAN1 or MCFD2 transcripts in NCBI and were screened with the Dual-Luciferase reporter assay. The optimal siRNAs were chemically modified and conjugated with three N-acetylgalactosamine molecules (GalNAc-siRNA), promoting their targeted delivery to the liver. The expression of LMAN1 and MCFD2 in cell lines or mice was examined by RT-qPCR and western blotting. For the mice administered with siRNA, we assessed their coagulation function by measuring APTT and the activity of FVIII factor. After administration, siRNAs GalNAc-LMAN1 and GalNAc-MCFD2 demonstrated effective and persistent LMAN1 and MCFD2 inhibition. 7 days after injection of 3mg/kg GalNAc-LMAN1, the LMAN1 mRNA levels reduced to 19.97% ± 3.78%. MCFD2 mRNA levels reduced to 32.22% ± 13.14% with injection of 3mg/kg GalNAc-MCFD2. After repeated administration, APTT was prolonged and the FVIII activity was remarkably decreased. The tail bleeding test of mice showed that the amount of bleeding in the treated group did not significantly increase compared with the control group. Our study confirms that therapy with RNAi targeting LMAN1-MCFD2 complex is effective and can be considered a viable option for anticoagulation drugs. However, the benefits and potential risk of bleeding in thrombophilic mice model needs to be evaluated.