Background: Xenotransplantation using pig cells, tissues, or organs may be associated with the transmission of porcine microorganisms and the development of zoonoses. Among all porcine microorganisms porcine endogenous retroviruses (PERVs) represent a special risk because they are integrated in the genome of all pigs and able to infect human cells. In previous preclinical and retrospective clinical trials of xenotransplantation, no transmission of PERV was observed. The first clinical trial of (alginate-encapsulated) porcine islet cell transplantation in New Zealand, which was approved by the New Zealand Government as an open-label phase I/IIa safety/efficacy trial, offers the possibility to analyze microbiological safety in a prospective clinical study. Methods: Before the trial started, a multilevel testing strategy was used to screen for 26 microorganisms in donor pigs of the Auckland Island strain and the islet cell preparations used for treatment. Donor testing was performed using molecular methods including multiplex real-time PCR. Blood samples from 14 pig islet cell recipients were also investigated by molecular biological methods at weeks 1, 4, 8, 12, 24, and 52 post-transplant for the transmission of porcine microorganisms. Sera were also monitored at these time points for antibodies against PERVs. Results: Beginning in 2009, fourteen patients with severe unaware hypoglycemia were treated with one of four different dosages of alginate-encapsulated porcine islets ranging from 5000-20 000 islet equivalents delivered in a single dose. No transmission of either PERVs or other porcine microorganisms was detected by PCR and immunological methods. Conclusion: These findings support previous results and strongly indicate the safety of xenotransplantation as performed here.

Chapter 2 of the original consensus statement published in 2009 by IXA represents an excellent basis for the production of safe donor pigs and pig-derived materials for porcine islet xenotransplantation. It was intended that the consensus statement was to be reviewed at interval to remain relevant. Indeed, many of the original salient points remain relevant today, especially when porcine islet xenotransplantation is performed in conjunction with immunosuppressants. However, progress in the field including demonstrated safe clinical porcine xenograft studies, increased understanding of risks including those posed by PERV, and advancement of diagnostic capabilities now allow for further consideration. Agents of known and unknown pathogenic significance continue to be identified and should be considered on a geographic, risk-based, dynamic, and product-specific basis, where appropriate using validated, advanced diagnostic techniques. PERV risk can be sufficiently reduced via multicomponent profiling including subtype expression levels in combination with infectivity assays. Barrier facilities built and operated against the AAALAC Ag Guide or suitable alternative criteria should be considered for source animal production as long as cGMPs and SOPs are followed. Bovine material-free feed for source animals should be considered appropriate instead of mammalian free materials to sufficiently reduce TSE risks. Finally, the sponsor retention period for archival samples of donor materials was deemed sufficient until the death of the recipient if conclusively determined to be of unrelated and non-infectious cause or for a reasonable period, that is, five to 10 yrs. In summary, the safe and economical production of suitable pigs and porcine islet xenograft materials, under appropriate guidance and regulatory control, is believed to be a viable means of addressing the unmet need for clinical islet replacement materials.

Previously a strategy for monitoring of pigs intended for cell transplantation was developed and successfully applied to several representative herds in New Zealand. A designated pathogen-free (DPF) herd has been chosen as a good candidate for xenotransplantation. This herd has previously tested free of infectious agents relevant to xenotransplantation and we present here an in depth study of porcine endogenous retrovirus (PERV) transmission. A panel of assays that describes the constraints for the transmission of PERV has been suggested. It includes a) infectivity test in coculture of DPF pig primary cells with both human and pig target cell lines; b) RT activity in supernatant of stimulated primary cells from DPF pigs; c) viral load in donor's blood plasma; d) PERV proviral copy number in DPF pig genome; e) PERV class C prevalence in the herd and its recombination potential.

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On behalf of DOL and LCT  A clinical trial in 14 adult type 1 diabetics intra‐peritoneal alginate microencapsulated neonatal porcine islets transplants commenced in NZ over 2 years ago and the last patient has just completed the required 12 months of follow up. To meet regulatory requirements for xenotransplantation, an elaborate program on xenovirology has been developed on one unique herd of pigs that was free from all conventional pathogens and was qualified as “null” pigs, i.e. they do not have a transmittable pig endogenous retrovirus (PERV). These pigs are the animal founders for the DPF donor herd. A comprehensive program for patients’ microbiology follow‐up was also developed. This program includes assays developed for monitoring potential infection with PERV and other potentially xenotic pathogens. All of the patients selected had unaware hypoglycaemia. The dose of islets used varied from 5000/kg (n = 4) 10 000/kg (n = 4) 15 000/kg (n = 4), and 20 000/kg (n = 2). The patients experience little if any side effects and there was no evidence of xenosis. Unaware hypoglycaemia was lessened in all groups but more so in the 10 000/kg group. Insulin dose was lowered and HbA1c improved in some but not all subjects. The patients with maximum insulin dose drop received 5000/kg dose. There was no clear cut relationship of return of hypoglycaemia awareness and glucagon or adrenergic responses to induced hypoglycaemia. There was direct laboratory evidence of islet beta cell function. Clinical follow up at 2 years of some patients indicates persistence of the benefits. A further clinical trial involving a different dosing schedule and some technical improvements is in progress. Choroid plexus (CP) ependymal epithelium secretes a wide variety of neurotrophic and neural support molecules. Micro‐encapsulated CP delivered intra‐cerebrally into rat models of stroke, Huntington's disease and Parkinson's disease show remarkable functional and histological recovery. In the quinolinic acid primate model of Huntington's and the MTPP model of Parkinson's functional recovery and relevant histological evidence of neuro‐regeneration were seen. The treatment was safe. A clinical trial of this form of treatment in humans with Parkinson's is being undertaken. Conclusions:  Xenotransplantation of microencapsulated islets and other cell types into human and non‐human primates from a biocertified herd of pigs prepared under GMP conditions can be conducted with safety and efficacy. Further improvements in clinical outcomes are expected with technical advances.

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