*Purpose: Porcine islet xenotransplantation offers a reliable and near limitless supply of islets for the treatment of type 1 diabetes. Intraportal infusion of cells exposes them to a highly inflammatory milieu, engaging molecular mechanisms leading to the nuclear translocation of the transcription factor, NFκB, and its transcriptional activity. Receptors that respond to acute inflammatory mediators and activate NFκB simultaneously initiate the machinery responsible for programmed cell death (PCD), which consists of two fundamental arms: caspase-dependent apoptosis and caspase-independent programmed necrosis. X-linked inhibitor of apoptosis, (XIAP) is considered a key cell death regulator including immune-mediated cell death due to its ability to regulate NFκB and suppress both mitochondrial and death-receptor activated apoptotic signaling. Therefore, we tested the hypothesis that if we overexpress XIAP in islets (before transplantation), we can suppress immune attack and enhance islet survival.

*Methods: We evaluated XIAP over-expression introduced in vitro via a plasmid or lentiviral vector in NPIs [wild type (wt) or transgenic with α1,3-galactosyltransferase knock-out (GalT-KO); human CD46 expressing]. Untransfected and transfected NPIs were cultured with rhesus monkey PBMC and serum to simulate in vivo transplant conditions. Western immunoblot, immunohistochemistry staining, viability and caspase 3/7 activity assays used to evaluate islet survival and function. In vivo transplantation of the control and XIAP-islets was carried out by a dual islet transplant model (DITM) procedure in Rhesus macaques that we have previously established.

*Results: Immunoblot, GFP imaging and staining demonstrated increased XIAP expression after XIAP plasmid transfection or lentiviral mediated infection in NPIs, and suppressed caspase activity (p=0.0057/48h; p=0.003, 72h; unpaired two-tailed t test) in islet cultures and under simulated co-culture conditions compared to uninfected (p=0.001 one way Anova). Data analysis of XIAP-islets in DITM are in progress.

*Conclusions: We have optimized an innovative co-culture assay to introduce isolated genetic modifications into NPIs and demonstrate their salutary effects. We have employed this technique in an in vivo model, thus making these modified NPIs available for head-to-head comparison with unmodified islets to gain insights into the relative utility of the transgene prior to full integration into a pig line.

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