Abstract
BACKGROUND: Pig xenografts offer a solution to human organ shortage; however, immune rejection is a major barrier. Immunomodulatory genes such as heme oxygenase-1 (HO1) and CD47 are key. Precise promoter control and strategic genomic integration for reliable expression and xenoantigen disruption are critical. Therefore, this study aimed to develop a promoter-pairing strategy for HO1 and CD47 in genetically modified pigs to achieve context-appropriate expression and enhance xenograft success. METHODS: We analyzed the transcriptional profiles of xenoantigens (GGTA1, B4GALNT2, and CMAH) in pig tissues using qPCR. Exon 4 of CMAH was selected for knock-in because of its low activity and xenoantigen role, enabling precise promoter-driven transgene expression and xenoantigen disruption. A dual-promoter cassette (inducible HO1 and constitutive CD47) was inserted into the CMAH locus of GGTA1-knockout fibroblasts using CRISPR/Cas9. The screened clones were used for somatic cell nuclear transfer to generate pigs. RESULTS: CMAH showed significantly lower and more consistent expression than did GGTA1/B4GALNT2 across tissues. In 293T cells and primary pig fibroblasts, HO1 was low at baseline but strongly induced by human serum/PMA, whereas CD47 exhibited high basal expression with inducibility. Cloned pigs with the HO1/CD47 cassette in the CMAH locus were successfully generated and validated. HO1 protein localized mainly to the liver and lungs, while CD47 was broadly expressed in tissues/blood leukocytes, confirming the tissue-specific and stimulus-responsive functions of the dual promoter. CONCLUSION: This study successfully established a promoter-optimized locus-specific knock-in strategy for inducing HO1 and constitutive CD47 in GGTA1-knockout pigs. This dual-promoter system enabled context-appropriate expression and enhanced xenograft compatibility. Future in vivo studies are crucial to evaluate long-term graft survival and HO1 inducibility, paving the way for clinical xenotransplantation.