Introduction: Dysregulated coagulation is a barrier to survival of pig organs in nonhuman primates. The protein C pathway is a key regulator of coagulation and inflammation. Thrombomodulin (THBD) binds circulating thrombin, converting it to an anticoagulant by changing its substrate specificity from prothrombin to protein C. EPCR promotes the activation of protein C by thrombin bound to THBD. We tried different gene-editing strategies to produce GTKO/SdaKO/CMAHKO/hCD55/hTHBD/hEPCR pigs.

Methods: We tested hEPCR gene knockin at various insertion sites in the pig genome, e.g., Rosa26, H11, COL1A1, and exon 9 of the GGTA1 gene. Different promoters, e.g., EF1α, CAG, pig ICAM2, and pig THBD, were used to express hEPCR at the COL1A1 site to evaluate expression. Three intron sequences of hEPCR were added to the coding sequence of hEPCR to test expression. hEPCR was connected with other transgenes by using 2A and IRES for detection.

Results: (1) Integration vectors of pig GGTA1 exon 9, Rosa26, H11, and COL1A1 were constructed by connecting the hEPCR gene with an EF1α promoter. The vector was transfected into pig fibroblasts, single colonies were analyzed to verify the EPCR knockin targeting site by PCR, and positive clones were incubated with fluorescently-labeled EPCR antibody to detect expression of hEPCR by flow cytometry. The four schemes resulted in no or only partial expression of hEPCR. (2) The hEPCR gene was connected with a CAG promoter and knocked-in at the COL1A1 site. Expression of hEPCR was almost completely suppressed. The pig endothelial-specific promoter ICAM2 was used to express hEPCR at the COL1A1 site. Cloned piglets were produced, but hEPCR expression in endothelial cells was very low (detected by FACS and IHC). (3) Three intron sequences of hEPCR were added into the coding sequence region, and the EF1α promoter was used to link these sequences and knockin to the COL1A1 site. Expression of hEPCR was suppressed. (4) A CAG-GFP-IRES-EPCR vector was knocked-in to the COL1A1 site. Expression of GFP and hEPCR was partially inhibited in the positive clones. (5) hTHBD and hEPCR linked by 2A were knocked-in to the COL1A1 site of GTKO/SdaKO/CMAHKO/hCD55 pigs under control of the pig endogenous THBD promoter. After cloned piglets were born, all vascular endothelial cells expressed both hTHBD and hEPCR well.

Conclusions: Transcriptional silencing of hEPCR in pig cells was similar when we used different insertion sites or promoters. The inhibition of GFP expression in the CAG-GFP-IRES-hEPCR assay showed that the inhibition occurred at the initiation of transcription. hEPCR was successfully expressed in all endothelial cells of GTKO/SdaKO/CMAHKO/hCD55/hTHBD/ hEPCR pigs, which we suggest was related to either or both of the following reasons: (1) The THBD promoter could not be inhibited. (2) The IRES and 2A generate hEPCR mRNA in different ways so that the residual amino acids after 2A cleavage possibly changed the spatial structure of the hEPCR protein.