Strong xenoprotective function by single-copy transgenes placed sequentially at a permissive locus.

BACKGROUND: Multiple xenoprotective transgenes are best grouped at a single locus to avoid segregation during breeding and simplify production of donor animals. METHODS: We used transgene stacking to place a human CD55 transgene adjacent to a human heme oxygenase 1 construct at the porcine ROSA26 locus. A transgenic pig was analyzed by PCR, RT-PCR, droplet digital PCR, immunohistochemistry, immunofluorescence, and flow cytometry. Resistance to complement-mediated cell lysis and caspase 3/7 activation were determined in vitro. RESULTS: The ROSA26 locus was retargeted efficiently, and animals were generated by nuclear transfer. RNA and protein analyses revealed abundant expression in all organs analyzed, including pancreatic beta cells. Transgenic porcine kidney fibroblasts were almost completely protected against complement-mediated lysis and showed reduced caspase 3/7 activation. CONCLUSION: Step-by-step placement enables highly expressed single-copy xenoprotective transgenes to be grouped at porcine ROSA26.

Engraftment and reversal of diabetes after intramuscular transplantation of neonatal porcine islet-like clusters.

BACKGROUND: Intraportal infusion is currently the method of choice for clinical islet cell transplantation but suffers from poor efficacy. As the liver may not represent an optimal transplantation site for Langerhans islets, we examined the potential of neonatal porcine islet-like clusters (NPICCs) to engraft in skeletal muscle as an alternative transplantation site. METHODS: Neonatal porcine islet-like clusters were isolated from 2- to 5-day-old piglets and either transplanted under the kidney capsule (s.k.) or injected into the lower hindlimb muscle (i.m.) of streptozotocin-diabetic NOD-SCID IL2rγ(-/-) (NSG) mice. Survival, vascularization, maturation, and functional activity were analyzed by intraperitoneal glucose tolerance testing and immunohistochemical analyses. RESULTS: Intramuscular transplantation of NPICCs resulted in development of normoglycemia and restored glucose homeostasis. Time to reversal of diabetes and glucose tolerance (AUC glucose and AUC insulin) did not significantly differ as compared to s.k. transplantation. Intramuscular grafts exhibited rapid neovascularization and graft composition with cytokeratin-positive ductal cells and beta cells at post-transplant weeks 2 and 8 and after establishment of normoglycemia was comparable in both groups. CONCLUSIONS: Intramuscular injection represents a minimally invasive but efficient alternative for transplantation of NPICCs and, thus, offers an attractive alternative site for xenotransplantation approaches. These findings may have important implications for improving the outcome and the monitoring of pig islet xenotransplantation.

Virus safety of islet cell transplantation from transgenic pigs to marmosets.

Transplantation of pig islet cells for the treatment of diabetes may be a more effective approach compared with the application of insulin. However, before introduction into the clinic, efficacy and safety of this treatment have to be shown. Non-human primate models may be used for this, despite the fact that they are characterised by several limitations. Here we investigate the prevalence of porcine endogenous retroviruses (PERVs), which are present in the genome of all pigs and which may infect human cells, as well as of porcine herpes viruses in donor pigs and their potential transmission to non-human primate recipients. Despite the fact that all three subtypes of PERV were present in all and porcine cytomegalovirus (PCMV) was found in some of the pigs, neither PERVs nor PCMV were found in the recipient animals under the experimental conditions applied. Porcine lymphotropic herpes viruses (PLHV) were not found in the donor pigs, hepatitis E virus (HEV) was not found in the recipients.