Introduction: Diabetes mellitus poses a significant global health burden, with islet transplantation recognized as a promising therapeutic strategy. However, the scarcity of human donors limits its widespread application. Porcine islet xenotransplantation offers a viable alternative, yet challenges persist regarding interspecies pathogen transmission and suboptimal glucose-stimulated insulin secretion (GSIS) efficiency compared to human islets. Here, we introduce the Xeno-1 pig line—a highly inbred, PERV-C-negative minipig breed developed through 20 generations of selective breeding. This study aims to characterize the genomic, physiological, and immunological attributes of Xeno-1 pigs and validate their safety and efficacy as donors for clinical porcine islet xenotransplantation.
Methods: ​Genetic Analysis: Whole-genome sequencing (WGS) of 90 Xeno-1 pigs was performed to assess genetic homogeneity and identify selective sweeps related to insulin secretion and glucose metabolism. Comparative genomic analyses were conducted against Duroc, Yorkshire, and Landrace breeds.
​PERV Characterization: PERV subtypes (A, B, C) were screened via genome sequencing and transcriptomic profiling across 12 tissues. Functional infectivity of PERV-C-like variants was evaluated using in vitro co-culture assays.
​Islet Function Assessment: RNA sequencing (RNA-seq) and GSIS assays were conducted on islets isolated from Xeno-1 and three control breeds (Duroc, Yorkshire, Landrace). Differential gene expression was analyzed using GO/KEGG enrichment and protein-protein interaction (PPI) network analysis.
​Microbiome Profiling: 16S rDNA sequencing and RNA-seq were used to compare gut microbial composition and immune-related gene expression in Xeno-1 pigs bred in designated pathogen-free (DPF) versus conventional environments.
​Clinical Trial: A pilot xenotransplantation trial was conducted in 10 type 1 diabetic patients using Xeno-1-derived islets, with long-term follow-up (up to 10 years) to assess safety and metabolic outcomes.
Results: ​Xeno-1 pigs exhibited a high inbreeding coefficient (F₂₀=0.986) and genome-wide homozygosity. Selective sweeps identified genes enriched in insulin secretion pathways (PDX1, PAX6, SLC30A8), glucose homeostasis (GYS2, CEBPA), and pancreatic development (NKX6-1). Xeno-1 pigs were PERV-C-negative, and PERV-A/C recombinant formation was inhibited. PERV-unclassified variants lacked intact viral genomes and failed to produce infectious particles in co-culture experiments. Xeno-1 islets demonstrated 2–4-fold higher GSIS compared to other controls (Duroc, Yorkshire, Landrace) (p<0.001). Key drivers included upregulated PDX1, PAX6, and SLC30A8 expression. DPF-reared Xeno-1 pigs showed reduced gut microbial diversity, with downregulated genes linked to adaptive immunity (LAG3, CD79A) and upregulated pathways involved in lipid catabolism. No PERV transmission or infections were detected in recipients over 10-year follow-up. Improved glycemic control was sustained, with reduced exogenous insulin requirements.
Conclusion: The Xeno-1 pig line exhibits genomic stability, enhanced islet function, and negligible PERV-related risks, establishing it as a safe and superior donor source for clinical porcine islet xenotransplantation. This study provides critical insights into optimizing donor pig breeding strategies and advancing the clinical translation of xenotransplantation therapies for diabetes.