Xenograft-Derived Cell-Free Deoxyribonucleic Acid as an Early Biomarker of Rejection in Genetically Engineered Pig-to-Non-Human Primate Kidney Xenotransplantation

Abstract

Introduction: Non-invasive biomarkers that detect xenograft injury before irreversible damage are essential for improving kidney xenotransplantation outcomes. This study investigated whether xenograft-derived cell-free DNA (xdcfDNA) is helpful as a non-invasive, early biomarker of antibody-mediated rejection.

Methods: Kidneys from genetically engineered pigs (GGTA1/CMAH/iGb3s/B4GalNT2 knockout; CD39, CD55, CD46, TBM knock-in) were transplanted into 10 cynomolgus monkeys, which received thymoglobulin, rituximab, anti-CD154 monoclonal antibody, corticosteroid, tacrolimus, and mycophenolate mofetil. Plasma xdcfDNA was measured using species-specific quantitative polymerase chain reaction. Pathological scoring and rejection diagnosis of the kidney xenograft biopsy were performed based on the Banff 2022 criteria.

Results: XdcfDNA levels increased markedly before an overt increase in serum levels of creatinine and blood urea nitrogen after kidney xenotransplantation. When recipients were classified into low- and high-score groups based on the Banff score of kidney xenograft biopsy, xdcfDNA levels were higher in the high-score groups for intimal arteritis (v), composite vasculitis (g + ptc + v), tubular atrophy (ct), interstitial fibrosis (ci), and IgG deposition. Furthermore, the rejection group showed higher xdcfDNA levels than the non-rejection group (p = 0.0270). The cut-off xdcfDNA value for xenograft rejection was 2.545%, with an apparent sensitivity of 100% (95% confidence interval, 64.57%-100.00%) and specificity of 100% (43.85%-100.00%).

Conclusions: XdcfDNA is a potentially sensitive, noninvasive, and early biomarker of xenograft rejection, capturing vasculitis and subsequent chronic injury. Periodic monitoring of xdcfDNA could support noninvasive screening of rejection before overt functional changes in xenografts emerge and might contribute to guiding confirmatory xenograft biopsy and subsequent immunosuppression modification.