Introduction: Corneal xenotransplantation, first attempted in the 19th century, was historically hindered by xenogeneic immune barriers. With the advent of alpha-1,3-galactosyltransferase gene-knockout pigs and CRISPR-Cas9-mediated multi-gene editing—including PERV inactivation—xenotransplantation has re-emerged as a viable clinical strategy. Given the persistent shortage of donor corneas in East Asia, porcine corneas are under investigation as alternative graft sources. While early genetically-engineered (GE) models showed limited graft survival without substantial immunosuppression, multi-gene-edited pigs are being developed to address this. However, the impact of extensive immune-related gene editing on corneal endothelial cell function remains unknown. This study assesses the biophysical integrity of endothelial cells in these multi-gene-edited porcine corneas, a critical determinant for their clinical applicability in full-thickness xenotransplantation.
Methods: Triple or quadruple knockout (T[Q]KO) pigs with targeted deletions in GGTA1, CMAH, β4GalNT2, and/or iGb3s were utilized, combined with knock-in (KI) or overexpression (OE) of human CD55, CD39, CD46, and thrombomodulin (TBM) genes. A total of 27 eyes from 14 genetically engineered pigs were analyzed to assess physical and functional suitability as corneal donors. Corneal endothelial cell density (ECD) was monitored over 7 days in storage medium. Endothelial cell proliferative capacity was assessed via doubling time (DT), and immunofluorescence was used to evaluate expression of endothelial pump and tight junction proteins.
Results: The GE pigs had a mean age of 11.9 months, with an average central corneal thickness of 718.2 μm. ECD loss was significantly greater in pigs under 6 months old (55.1%) compared to those over 6 months (8.8%) (p<0.001). T(Q)KO/hCD46OE/hTBMOE corneas exhibited early endothelial apoptosis or necrosis, whereas other GE corneas did not show such changes. Although DT was increased across all GE pigs, there was no age-related difference. Key endothelial markers (ZO-1, N-cadherin, ATPase, SLC4A1, and aquaporin 1) were consistently expressed across all GE variants.
Conclusion: These findings suggest that corneas from both T(Q)KO/hCD55KI/hCD39KI and QKO pigs over 6 months of age exhibit sufficient endothelial cell function, making them suitable donor candidates for human corneal xenotransplantation.