Introduction: Post-transplant anemia (PTA) is a frequent complication observed in 40% of allogeneic kidney transplant recipients. The erythropoietin (EPO) produced by the kidney is a glycoprotein that stimulates the production of red blood cells, the molecular incompatibilities between pig and human EPO may lead to the aggravation of anemia after porcine to human transplantation. Genetic modification of donor kidneys to over-express human EPO may offer a novel therapeutic strategy for ameliorating PTA in transplant recipients.
Methods: Based on GTKO/CD55 pigs, the CRISPR/Cas9 system was utilized to insert an expression vector into the COL1A1 locus via homologous recombination, generating transgenic pigs with endothelial cell-specific expression of human EPO. Serum from the pigs was isolated and analyzed by ELISA to quantify human-derived EPO levels. Primary endothelial cells were also isolated from the pigs for expression validation, with detecting human EPO level in the cell culture supernatant. Subsequently, pig kidneys were transplanted into rhesus monkeys to monitor post-transplant hemoglobin levels and comparative assessment of functional maintenance between organ-intrinsic EPO expression and exogenous rhEPO administration.
Results: By genetic editing of GTKO/CD55 pig fibroblasts, six positive cell clones were obtained. Through somatic cell nuclear transfer (SCNT), five piglets expressing human EPO were ultimately generated. Human EPO was not detected in wild pig serum, compared to human serum (6.90 mIU/mL), elevated levels of human-derived EPO were detected in the serum of EPO transgenic pig serum (78.23 mIU/mL) . Porcine endothelial cells were isolated, and human EPO could also be detected in the culture supernatant (20 mIU/mL). In pig-to-monkey kidney transplantation experiments, two recipient monkeys transplanted with EPO transgenic pig kidneys could maintain relatively stable hemoglobin content within 3 weeks without the exogenous human EPO injection. However, the hemoglobin of those transplanted with GTKO/βGalKO/CD55/TM kidneys gradually decreased one week after transplantation, and exogenous rhEPO treatment was necessary. Furthermore, we observed that EPO-transgenic pigs exhibited significantly higher red blood cell counts and hemoglobin levels compared to normal piglets, demonstrating that human-derived EPO can promotes erythropoiesis in pigs.
Conclusion: Donor kidneys with human EPO transgene can produce high-level human-derived EPO in pig serum and have demonstrated the potential in alleviating post-transplant anemia, but data was constrained by the limited groups and short observing time, necessitating further investigation in the future.