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Genetically engineered pigs are now available for xenotransplantation in which all three known carbohydrate xenoantigens, against which humans have natural antibodies, have been deleted (triple-knockout [TKO] pigs). Furthermore, multiple human transgenes have been expressed in the TKO pigs, all of which are aimed at protecting the cells from the human immune response. Many human sera demonstrate no or minimal antibody binding to, and little or no cytotoxicity of, cells from these pigs, and this is associated with a relatively low T-cell proliferative response. Unfortunately, baboons and other Old World NHPs have antibodies against TKO pig cells, apparently directed to a fourth xenoantigen that appears to be exposed after TKO. In our experience, most, if not all, humans do not have natural antibodies against this fourth xenoantigen. This discrepancy between NHPs and humans is providing a hurdle to successful translation of pig organ transplantation into the clinic, and making it difficult to provide pre-clinical data that support initiation of a clinical trial. The potential methods by which this obstacle might be overcome are discussed. We conclude that, whatever currently available genetically engineered pig is selected for the final pre-clinical studies, this may not be the optimal pig for clinical trials.

During the last years, progress has been made in survival and function of pig-to-non-human primate organ xenotransplantation using organs from genetically modified pigs and immunosuppression regimens that are clinically acceptable. This, together with increased insights into a low risk of pig-to-human transmission of porcine endogenous retrovirus, has opened the perspective of starting with first-in-human trials with xenogeneic organs. The regulatory path to clinical development is complex. Unlike an organ from human donors, an organ from pigs, either genetically modified or wild-type pigs, is considered a medicinal product for human use and hence is under regulatory oversight, in the United States by the Food and Drug Administration and in Europe by the national competent authorities of the member states as well as the European Medicines Agency. Related to the status of medicinal product, "(current) good practices" apply in the process of generating a xenogeneic organ through to the transplantation into a patient and life-long follow-up. In addition, guidances for xenotransplantation products and genetically modified organisms do apply as well. This commentary focuses on regulatory aspects of transplantation of organs from genetically modified pigs into humans, with the intention to facilitate the interactions between regulatory agencies and institutions (sponsors) in research and clinical development of these organs, to support the perspective of speeding up the process with a proper entry in clinical application, to fill an unmet medical need in patients with end-stage organ disease.

The main benefit of xenotransplantation is its potential to overcome the worldwide organ shortage experienced in allotransplantation. Allogeneic transplantation is the only successful therapy for several life-threatening diseases, with cell, tissue or organ donation only partially meeting the demand and many patients dying while waiting for treatment. With supply falling short of demand, it is foreseen that the use of porcine material may at some stage overcome the existing gap between organ availability and clinical need. Recently, pig islet cells have been utilised in clinical trials, with safety being demonstrated. Indeed, pig-derived cells present several advantages: i) porcine cells have a stable function and differentiation pattern and are not tumorigenic; ii) pig cells have been shown to meet the physiological needs in large animal models; iii) the source of pig cells can be scaled up to meet demands in a highly standardised manner, and with respect to animal welfare regulations; iv) 'designated-pathogen-free' (DPF) pig lines can be produced, which could result in a higher safety profile than allotransplantation itself; v) the risk of zoonosis, which was raised years ago as the major hurdle, has been recently circumvented and is actually viewed as a controlled risk; and vi) immune risks are being circumvented via the use of genetically modified donor animals and encapsulation of porcine cells, particularly for the treatment of diabetes. Overall, the benefit appears to outweigh potential risks with respect to cellular xenotransplantation and this is discussed further in this review.

This review presents an overview on the present status of xenogeneic islet cell transplantation. In four sections we present (1) a short introduction on clinical islet transplantation using islets from deceased humans, ending with the rationale for xenogeneic islet transplantation; (2) porcine islet survival and function in diabetic nonhuman primates; (3) features in this animal model that are relevant for clinical development; and (4) limitations and translational value of the model in nonhuman primates.

A series of immunosuppressives is nowadays on the market or in advanced clinical development that are efficacious in prevention or treatment of rejection of a transplant in patients with end-stage organ failure. These can roughly be divided into lowmolecular-weight XENOBIOTICS, orally active drugs produced by microorganisms or chemical synthesis, and biologicals, (monoclonal) antibodies or rDNA fusion proteins. Most XENOBIOTICS work intracellularly and affect different pathways in lymphocyte activation and/or proliferation; since such pathways are not truly selective for LYMPHOCYTES, most of these drugs show inherent side-effects and generally have a low therapeutic window. However, the availability of novel agents with a broader therapeutic window, and refinements in combination treatment have greatly added to improved tolerability of immunosuppressive regimens. Broadly acting cytotoxic drugs with severe side-effects are gradually being replaced by compounds with a more selective action towards lymphoid cells, and corticosteroid-sparing regimens are being applied to reduce the adverse side-effects of corticosteroids. Most biologicals work by binding to cell surface molecules, resulting in inactivation or depletion of the target cells. Progress in this area has not only resulted in improved IMMUNOSUPPRESSION, but also in potential approaches to induce a status of unresponsiveness, i.e., TOLERANCE to the transplant. This is achieved either by efficient depletion of reactive cells or by inhibition of second signals in coreceptor blockade in T CELL activation. New paradigms in IMMUNOSUPPRESSION involve interference with cell trafficking (lymphocyte recirculation) that appears possible by using either innovative XENOBIOTICS or biologicals binding to cell ADHESION MOLECULES.

Background We established a Source Animal (barrier) Facility (SAF) for generating designated pathogen‐free (DPF) pigs to serve as donors of viable organs, tissues, or cells for xenotransplantation into clinical patients. This facility was populated with caesarian derived, colostrum deprived (CDCD) piglets, from sows of conventional‐specific (or specified) pathogen‐free (SPF) health status in six cohorts over a 10‐month period. In all cases, CDCD piglets fulfilled DPF status including negativity for porcine circovirus (PCV), a particularly environmentally robust and difficult to inactivate virus which at the time of SAF population was epidemic in the US commercial swine production industry. Two outbreaks of PCV infection were subsequently detected during sentinel testing. The first occurred several weeks after PCV‐negative animals were moved under quarantine from the nursery into an animal holding room. The apparent origin of PCV was newly installed stainless steel penning, which was not sufficiently degreased thereby protecting viral particles from disinfection. The second outbreak was apparently transmitted via employee activities in the Caesarian‐section suite adjacent to the barrier facility. In both cases, PCV was contained in the animal holding room where it was diagnosed making a complete facility depopulation‐repopulation unnecessary. Method Infectious PCV was eliminated during both outbreaks by the following: euthanizing infected animals, disposing of all removable items from the affected animal holding room, extensive cleaning with detergents and degreasing agents, sterilization of equipment and rooms with chlorine dioxide, vaporized hydrogen peroxide, and potassium peroxymonosulfate, and for the second outbreak also glutaraldehyde/quaternary ammonium. Impact on other barrier animals throughout the process was monitored by frequent PCV diagnostic testing. Result After close monitoring for 6 months indicating PCV absence from all rooms and animals, herd animals were removed from quarantine status. Conclusion Ten years after PCV clearance following the second outbreak, due to strict adherence to biosecurity protocols and based on ongoing sentinel diagnostic monitoring (currently monthly), the herd remains DPF including PCV negative.

The treatment of acute liver failure, a condition with high mortality, comprises optimal clinical care, and in severe cases liver transplantation. However, there are limitations in availability of organ donors. Hepatocyte transplantation is a promising alternative that could fill the medical need, in particular as the bridge to liver transplantation. Encapsulated porcine hepatocytes represent an unlimited source that could function as a bioreactor requiring minimal immunosuppression. Besides patients with acute liver failure, patients with alcoholic hepatitis who are unresponsive to a short course of corticosteroids are a target for hepatocyte transplantation. In this review we present an overview of the innate immune barriers in hepatocyte xenotransplantation, including the role of complement and natural antibodies; the role of phagocytic cells and ligands like CD47 in the regulation of phagocytic cells; and the role of Natural Killer cells. We present also some illustrations of physiological species incompatibilities in hepatocyte xenotransplantation, such as incompatibilities in the coagulation system. An overview of the methodology for cell microencapsulation is presented, followed by proof-of-concept studies in rodent and nonhuman primate models of fulminant liver failure: these studies document the efficacy of microencapsulated porcine hepatocytes which warrants progress towards clinical application. Lastly, we present an outline of a provisional clinical trial, that upon completion of preclinical work could start within the upcoming 2-3 years.

BackgroundThe manufacturing process of islets includes a culture step which was originally introduced to ease the logistics of procedures in preparing the graft and transplant recipient. It has been suggested that culture at room temperature has an advantage over culture at 37 degrees C, in part by reducing immunogenicity via preferential elimination of contaminating cells (such as passenger leukocytes) within islets. We investigated this using islets isolated from pancreata of adult pigs. MethodsPorcine islets were isolated from three donors and cultured at 37 degrees C for 1day, and then under three different conditions: 37 degrees C for 6days (condition A); 22 degrees C for 6days (condition B); or 22 degrees C for 5days followed by 37 degrees C for 1day (condition C). Recovery was assessed by DNA measurement, viability by oxygen consumption rate normalized for DNA (OCR/DNA), and gene expression by RT-PCR for a series of 9 lymphocyte markers, 11 lymphokines and chemokines, and 14 apoptotic and stress markers. ResultsPost-culture islet recoveries were similar for the three culture conditions. Average OCR/DNA values were 129-159nmol/minmgDNA before culture, and 259-291, 204-212, and 207-228nmol/minmgDNA, respectively, for culture under conditions A, B, and C, respectively. Irrespective of culture condition, examined gene expression in all three series of lymphocyte markers, lymphokines and chemokines, and apoptotic and stress markers manifested a statistically significant decrease upon culture for 7days. This decrease was most dramatic for condition A: in particular, most of lymphocyte markers showed a >10-fold reduction and also six markers in the lymphokine and chemokine series; these reductions are consistent with the elimination of immune cells present within islets during culture. The reduction was less for apoptotic and stress markers. For culture under condition B, the reduction in gene expression was less, and culture under condition C resulted in gene expression levels similar to those under condition A: this indicates that 24h at 37 degrees C is sufficient to re-equilibrate gene expression levels from those in islets cultured at 22 degrees C to those in islets cultured at 37 degrees C. Results were consistent among the preparations from the three donors. ConclusionsCulture of porcine islets at 37 degrees C provides benefits over culture at 22 degrees C with respect to OCR/DNA outcomes and reduced expression of genes encoding lymphocyte markers, lymphokines and chemokines, and markers for apoptosis and stress.

Worldwide, there is a constant rise in the number of patients with end-stage organ failure in critical need for transplants, but the number of organs/cells available from deceased or living human donors is limited. Xenotransplantation using pig organs/tissues repre-sents a potential solution for this shortage; however, it has been hampered by a number of mainly immuno-logical hurdles. Remarkable progress was presented at the latest biennial (13th) international congress of the International Xenotransplantation Association, November 2015 in Melbourne, Australia, and the American Transplant Congress, May 2016 in Boston, USA. Most importantly, the survival records of pig organ xenografts in nonhuman primate models have strikingly improved with the use of multitransgenic pigs. Moreover, no safety issues were encountered in clinical trials with porcine islets, and the removal of porcine endogenous retroviruses from the genome of a pig cell line by the CRISPR/Cas9 technology offers the perspective to overcome the perceived potential risk of xenozoonosis in the near future. For all these reasons, interest in xenotransplantation has been boosted. This review summarises the current status of xenotransplantation research, including Swiss contri-butions as well as regulatory and safety aspects in the light of upcoming clinical trials.

Porcine islet xenotransplantation is considered a potential cell-based therapy for type 1 diabetes. It is currently being evaluated in diabetic nonhuman primates (NHP) to assess safety and efficacy of the islet product. However, due to a variety of distinct differences between the respective species, including the insulin secretory characteristics of islets, the suitability and predictive value of the preclinical model in the extrapolation to the clinical setting remain a critical issue. Islets isolated from human (n = 3), NHP (n = 2), adult pig (AP, n = 3), and juvenile pig (JP, n = 4) pancreata were perifused with medium at basal glucose (2.5 mm) followed by high glucose (16.7 mm) concentrations. The total glucose-stimulated insulin secretion (GSIS) was calculated from generated insulin secretion profiles. Nonhuman primate islets exhibited GSIS 3-fold higher than AP islets, while AP and JP islets exhibited GSIS 1/3 and 1/30 of human islets, respectively. The insulin content of NHP and AP islets was similar to that of human islets, whereas that of JP islets was 1/5 of human islets. Despite the fact that human, NHP, and AP islets contain similar amounts of insulin, the much higher GSIS for NHP islets than for AP and JP islets suggests the need for increased dosing of islets from JP and AP in pig-to-NHP transplantation. Porcine islet xenotransplantation to humans may require significantly higher dosing given the lower GSIS of AP islets compared to human islets.

Background: Various groups have reported prolonged diabetes reversal and graft function after porcine islet transplantation into diabetic macaques using different experimental designs (macaque source, islet source, type of immunosuppression): subsequently, the International Xenotransplantation Association has published recommendations for entering a clinical trial. Our experiments showed limitations that affected consistent achievement of long-term survival. We aimed to identify these limitations and underlying causes to emphasize the translational value of this highly relevant type 1 diabetic macaque model. Methods: We reviewed data from our institution and literature data on long-term porcine islet xenograft survival in the diabetic macaque model, especially focusing on aspects of incomplete diabetes reversal relative to macaque normal values. This phenomenon was compared with diabetes reversal in an allo-islet transplant model in macaques and with chronic insulin treatment of diabetic macaques, all with 180-day follow-up. This comparison enabled to identify potential model limitations and underlying causative factors. Results: Especially in the xenograft model, the achievement of long-term graft survival revealed limitations including chronic, mild hyperglycemia and absence of body weight (BW) gain or even progressive BW loss. Metabolic incompatibilities in glycemic control (i.e., insulin kinetics) between the pig and macaque species underlie chronic, mild hyperglycemia. This phenomenon might not bear relevance for the pig-to-human species combination because the glycemic control in pigs and humans is similar and differs from that in nonhuman primates (NHP). Weight loss could be related to changes in the gastrointestinal tract related with local high exposure to orally administered immunosuppressants; these must be given at higher dose levels because of low bioavailability in macaques to achieve systemic exposure at therapeutic levels. This is aggravated by insufficient graft insulin production in proportion to the needs of macaques: this model limitation has no translational value to the pig-to-human setting. Nutritional deficits can result in incorrect interpretation of blood glucose levels and C-peptide levels regarding graft function. Likewise, nutritional status alters physiologic responses, influencing susceptibility to infectious and noninfectious complications. Conclusion: The model-induced confounding described interferes with accurate interpretation of safety and efficacy studies, which affects the translational value of pig-to-NHP islet cell transplant studies to the pig-to-human transplant condition.

Clinical islet transplantation using human pancreasdonors in patients with type 1 diabetes mellitus isan established cellular therapy, primarily, for thosepatients who suffer from severe hypoglycemicunawareness [1]. However, the low availability ofpancreata that are suitable for islet manufacturingand the variability in isolated human islet qualityhinders this treatment from becoming broadlyapplicable to meet the medical need. NeonatalKitzmann JP, Law L, Shome A, Muzina M, Elliott RB, Mueller KR,Schuurman H-J, Papas KK. Real-time assessment of encapsulatedneonatal porcine islets prior to clinical xenotransplantation.Xenotransplantation 2012; 19: 333–336. 2012 John Wiley & Sons A/S.Abstract: Background: Porcine islet transplantation is emerging as anattractive option for the treatment of patients with type 1 diabetes, withthe possibility of providing islets of higher and more consistent qualityand in larger volumes than available from human pancreata. The use ofencapsulated neonatal porcine islets (ENPI) is appealing because it canaddress islet supply limitations while reducing the need for anti-rejectiontherapy. Pre-transplant characterization of ENPI viability and potencyis an essential component of the production process. We applied thevalidated assay for oxygen consumption rate normalized for DNAcontent (OCR/DNA) to characterize ENPI viability.Methods: ENPI of low viscosity and high m alginate were preparedaccording to standard methods and characterized at various culture timepoints up to 5 weeks.Results: The OCR/DNA (nmol/minAEmgDNA ± SEM) of ENPI(235 ± 10, n = 9) was comparable to that of free NPI (255 ± 14,n = 13). After encapsulation, NPI OCR/DNA was sustained over aculture period of up to 5 weeks. The average OCR/DNA of ENPIcultured longer than 9 days was higher than that of freshly encapsulatedNPI.Conclusion: This is the first characterization of ENPI by a validated andmore sensitive method for product viability. The NPI encapsulationprocess does not compromise viability as measured by OCR/DNA, andENPI can be cultured for up to 5 weeks with maintenance of viability.ENPI meet or exceed current adult porcine islet product release criteria(established at the University of Minnesota) for preclinical xenotrans-plantation in terms of OCR/DNA.

Introduction—Streptozotocin-induced diabetic nonhuman primates are used to study efficacy and safety of innovative immunosuppression after islet transplantation. We implemented a training program for medical management of a chronic disease state. Methods—Cooperation with hand feeding and drinking; shifting; and limb presentation were trained utilizing predominately positive but also negative reinforcement in 52 animals compared with 28 macaques subjected to conventional physical and/or chemical restraint. The success of and timing of behavior acquisition was evaluated in a representative subset of 14 animals. Results—Over 90% of animals were successful in behavior acquisition. Programmatically this resulted in complete elimination of chair restraint and negligible requirement for sedation. About half of trained animals had no to moderate thymic involution, indicative of a substantial reduction in stress.

Suszynski TM, Rizzari MD, Kidder LS, Mueller K, Chapman CS, Kitzmann JP, Pongratz RL, Cline GW, Todd PW, Kennedy DJ, O’Brien TD, Avgoustiniatos ES, Schuurman H-J, Papas KK. Paramagnetic microparticles do not elicit islet cytotoxicity with co-culture or host immune reactivity after implantation. Xenotransplantation 2011; 18: 239–244. © 2011 John Wiley & Sons A/S. Abstract: Background: Paramagnetic microparticles (MPs) may be useful in pancreatic islet purification, in particular purification of porcine islets as a potential xenotransplantation product. We assessed whether MPs affect islet function or induce an adverse effect following implantation. Methods: Porcine islets were co-cultured with 0, 500, and 1500 MPs per islet equivalent (IE) for 1 day and with 0 and 1500 MPs/IE for 7 days. Fractional viability was assessed using oxygen consumption rate normalized to DNA content (OCR/DNA) and after 7-day co-culture by perifusion glucose-stimulated insulin secretion (GSIS) and by transplantation under the renal capsule of diabetic nude mice. To assess an inflammatory response or immune reaction, MPs (∼107) were implanted under the renal capsule of C57BL/6 mice. Results: No statistically significant differences were measured in OCR/DNA (mean ± SE) following 1-day co-culture with 0, 500, or 1500 MPs/IE (243.3 ± 4.5, 211.3 ± 8.1, or 230.6 ± 11.3 nmol/min·mgDNA, respectively) or following 7-day co-culture with 0 or 1500 MPs/IE (248.5 ± 1.4 or 252.9 ± 4.7 nmol/min·mgDNA, respectively). GSIS was not affected by the presence of MPs; first- and second-phase insulin area-under-the-curve (mean ± SE) reflected no statistically significant differences after 7-day co-culture between 0 and 1500 MPs/IE (8.36 ± 0.29 and 8.45 ± 0.70 pg/ml·min·ngDNA for first-phase; 69.73 ± 2.18 and 65.70 ± 4.34 pg/ml·min·ngDNA for second-phase, respectively). Islets co-cultured with MPs normalized hyperglycemia in diabetic nude mice, suggesting no adverse effects on in vivo islet function. Implantation of MPs did not elicit tissue injury, inflammatory change or immune reactivity. Conclusion: MPs do not adversely affect islet viability or function during co-culture, and MPs are not immune reactive following implantation.

After feedback from the working parties, the final session focused on drafting proposed revisions of the WHO documents, and resulted in the formulation of the draft “Third WHO Global Consultation on Regulatory Requirements for Xenotransplantation Clinical Trials, The 2018 Changsha Communiqué.” This draft was submitted to WHO in February 2019 for WHO and World Health Assembly consideration. If approved, the 2018 Changsha Communiqué will then be posted on the websites of WHO, IXA, and TTS, and published in Xenotransplantation. This report includes summaries of the various sessions, followed by the abstracts of invited speakers from the update sessions.

Abrahante JE, Martins K, Papas KK, Hering BJ, Schuurman H-J, Murtaugh MP. Microbiological safety of porcine islets: comparison with source pig. Xenotransplantation 2011; 18: 88–93. © 2011 John Wiley & Sons A/S. Abstract: Background: Pig islet donors intended for clinical xenotransplantation for the treatment of diabetes must meet stringent conditions. Among others, viruses with the potential to cross the species barrier should be excluded from the herd: this list includes encephalomyocarditis virus (EMCV), hepatitis E virus (HEV), porcine cytomegalovirus (PCMV) and porcine γ-lymphotropic herpesvirus (PLHV). As an islet product is isolated from the pancreas and then subjected to culture before implantation, the question is raised whether islets could be negative even if the animal itself is positive for a distinct pathogen. Methods: To answer this question, sensitive quantitative real-time PCR assays were established for EMCV, HEV, PCMV and PLHV. Twelve adult animals from a high-hygienic herd were then evaluated; testing tissues, where the virus is expected to reside in latent infection, testing islets immediately after isolation, and then isolated islets after a 7-day culture. Results: None of the tissues tested positive for EMCV, HEV or PLHV. PCMV was observed in spleen tissue from six animals: three of these six animals were positive for isolated islets, and two of these three cases were also positive for islets after culture. Older animals in particular showed positivity, and within a given litter not all animals were PCMV positive. Conclusions: These data fit with spread through the herd by horizontal transmission, not in utero infection. PCMV has to be excluded from the herd to ensure that islets for transplantation are negative for PCMV.

Nguyen B-NH, Azimzadeh AM, Schroeder C, Buddensick T, Zhang T, Laaris A, Cochrane M, Schuurman H-J, Sachs DH, Allan JS, Pierson RN. Absence of Gal epitope prolongs survival of swine lungs in an ex vivo model of hyperacute rejection. Xenotransplantation 2011; 18: 94–107. © 2011 John Wiley & Sons A/S. Abstract: Background: Galactosyl transferase gene knock-out (GalTKO) swine offer a unique tool to evaluate the role of the Gal antigen in xenogenic lung hyperacute rejection. Methods: We perfused GalTKO miniature swine lungs with human blood. Results were compared with those from previous studies using wild-type and human decay-accelerating factor-transgenic (hDAF+/+) pig lungs. Results: GalTKO lungs survived 132 ± 52 min compared to 10 ± 9 min for wild-type lungs (P = 0.001) and 45 ± 60 min for hDAF+/+ lungs (P = 0.18). GalTKO lungs displayed stable physiologic flow and pulmonary vascular resistance (PVR) until shortly before graft demise, similar to autologous perfusion, and unlike wild-type or hDAF+/+ lungs. Early (15 and 60 min) complement (C3a) and platelet activation and intrapulmonary platelet deposition were significantly diminished in GalTKO lungs relative to wild-type or hDAF+/+ lungs. However, GalTKO lungs adsorbed cytotoxic anti-non-Gal antibody and elaborated high levels of thrombin; their demise was associated with increased PVR, capillary congestion, intravascular thrombi and strong CD41 deposition not seen at earlier time points. Conclusions: In summary, GalTKO lungs are substantially protected from injury but, in addition to anti-non-Gal antibody and complement, platelet adhesion and non-physiologic intravascular coagulation contribute to Gal-independent lung injury mechanisms.

Islet cell transplantation in nonhuman primates is generally performed in the liver, by infusion of the transplant into the portal vein. We introduced a vascular access port with the catheter tip located in the splenic vein to avoid multiple major survival surgeries. This procedure was conducted in 16 cynomolgus and 9 rhesus macaques. A subset underwent islet cell transplantation. A historic control group (n = 17) received the transplant via open midline laparotomy. The groups did not differ in operation time (median about 60 min): however, animals undergoing midline laparotomy required significantly more opioid pain relief postoperatively than animals implanted with a hepatic vascular access port. Animals after port placement and transplantation had significantly higher blood hemoglobin values than those in the control group, but these values were still in the normal range. In addition to transplantation, the port could be used for administration of biologics and for blood sampling. In all cases, the port remained patent for infusion purposes (median follow-up 336 days, range 62-485 days). Patency for blood sampling was maintained in about half of the animals: the 50% survival of patency for sampling was 255 days. This difference between infusion and sampling patency is most likely due to the location of the catheter tip in the splenic vein, with occlusion caused by the small vessel-to-catheter ratio. We conclude that hepatic vascular access enables long-term frequent administration of cells, medication, or other products and also serves to sample blood: hence, this procedure contributes to a higher level of animal's well-being.

Islet cell transplantation is in clinical development for type 1 diabetes. There are no data on the cost in relationship to its benefits. We performed a cost-effectiveness analysis and made a comparison with standard insulin therapy, using Markov modeling and Monte Carlo simulations. The patient population was adults aged 20 yr suffering from hypoglycemia unawareness. Data were estimates from literature and clinical trials: costs were based on the situation in the United States. For insulin therapy, cumulative cost per patient during a 20-yr follow-up was $663,000, and cumulative effectiveness was 9.3 quality-adjusted life years (QALY), the average cost-effectiveness ratio being $71,000 per QALY. Islet transplantation had a cumulative cost of $519,000, a cumulative effectiveness of 10.9 QALY, and an average cost-effectiveness ratio of $47,800. During the first 10 yr, costs for transplantation were higher, but cumulative effectiveness was higher from the start onwards. In sensitivity analyses, the need for one instead of two transplants during the first year did not affect the conclusions, and islet transplantation remained cost-saving up to an initial cost of the procedure of $240,000. This exploratory evaluation shows that islet cell transplantation is more effective than standard insulin treatment, and becomes cost-saving at about 9-10 yr after transplantation.

Porcine hepatocytes transplanted during acute liver failure might support metabolic functions until the diseased liver recovers its function. Here, we isolated high numbers of viable pig hepatocytes and evaluated hepatocyte functionality after encapsulation. We further investigated whether coculture and coencapsulation of hepatocytes with human multipotent mesenchymal stromal cells (MSC) are beneficial on hepatocyte function. Livers from 10 kg pigs (n=9) were harvested, and hepatocytes were isolated from liver suspensions for microencapsulation using alginate and poly(ethylene-glycol)- (PEG-) grafted alginate hydrogels, either alone or in combination with MSC. Viability, albumin secretion, and diazepam catabolism of hepatocytes were measured for one week. 9.2 ± 3.6 × 109hepatocytes with 95.2 ± 3.1% viability were obtained after isolation. At day 3, free hepatocytes displayed 99% viability, whereas microencapsulation in alginate and PEG-grafted alginate decreased viability to 62% and 48%, respectively. Albumin secretion and diazepam catabolism occurred in free and microencapsulated hepatocytes. Coencapsulation of hepatocytes with MSC significantly improved viability and albumin secretion at days 4 and 8 (p<0.05). Coculture with MSC significantly increased and prolonged albumin secretion. In conclusion, we established a protocol for isolation and microencapsulation of high numbers of viable pig hepatocytes and demonstrated that the presence of MSC is beneficial for the viability and function of porcine hepatocytes.

Beckwith J, Nyman JA, Flanagan B, Schrover R, Schuurman H-J. A health-economic analysis of porcine islet xenotransplantation. Xenotransplantation 2010; 17: 233–242. © 2010 John Wiley & Sons A/S. Abstract: Background: Islet cell transplantation is a promising treatment for type 1 diabetes. To overcome the shortage of deceased human pancreas donors, porcine islet cell xenotransplantation is being developed as an alternative to allotransplantation. The objective of this study was to perform a cost-effectiveness analysis of porcine islet transplantation in comparison with standard insulin therapy. The patient population for this study was young adults, ages 20 to 40, for whom standard medical care is inadequate in controlling blood glucose levels (hypoglycemia unawareness). Since trial data were lacking, estimates used extrapolations from data found in the literature and ongoing trials in clinical allotransplantation. Cost estimates were based on the data available in the USA. Methods: Markov modeling and Monte Carlo simulations using software specifically developed for health-economic evaluations were used. Outcomes data for ongoing clinical islet allotransplantation from the University of Minnesota were used, along with probabilities of complications from the Diabetes Control and Complications Trial. Quality-adjusted life years (QALYs) were the effectiveness measure. The upper limit of being cost-effective is $100 000 per QALY. Cost data from the literature were used and adjusted to 2007 US dollars using the medical care portion of the Consumer Price Index. Results: In both Markov modeling and Monte Carlo simulations, porcine islet xenotransplantation was both more effective and less costly over the course of the 20-yr model. For standard insulin therapy, cumulative cost per patient was $661 000, while cumulative effectiveness was 9.4 QALYs, for a cost of $71 100 per QALY. Transplantation had a cumulative cost of $659 000 per patient, a cumulative effectiveness of 10.9 QALYs, and a cost per QALY of $60 700. Islet transplantation became cost-effective at 4 yr after transplantation, and was more cost-effective than standard insulin treatment at 14 yr. These findings are related to relative high costs in the transplantation arm of the evaluation during the first years while those in the insulin arm became higher later in follow-up. Throughout the follow-up period, effectiveness of transplantation was higher than that of insulin treatment. In sensitivity analysis, duplication or triplication of one-time initial costs such as costs of donor animal, islet manufacturing and transplantation had no effect on long-term outcome in terms of cost-saving or cost-effectiveness, but the outcome of transplantation in terms of diabetes complications in cases with partial graft function could affect cost-saving and cost-effectiveness conclusions. Conclusion: Despite limitations in the model and lack of trial data, and under the assumption that islet transplantation outcomes for young adult type 1 diabetes patients are not dependent on the source of islet cells, this health-economic evaluation suggests that porcine islet cell xenotransplantation may prove to be a cost-effective and possibly cost-saving procedure for type 1 diabetes compared to standard management.

Xenotransplantation using porcine cells, tissues, or organs may offer a potential solution for the shortage of allogeneic human organs. Prior to the clinical use of porcine xenotransplants, three main hurdles must be overcome: immunologic rejection, physiologic incompatibility, and risk of transmission of porcine pathogens. Designated pathogen-free breeding of pigs can prevent transmission of most porcine microbes. However, this is not possible in the case of porcine endogenous retroviruses (PERV), which are integrated in the pig genome and can infect human cells in vitro. In order to assess the probability of transmission of PERV, a careful screening of the source pig herd is recommended. Two types of PERV are present in pigs, human-tropic PERV-A and PERV-B, which are both present in the genome of all pigs, and PERV-C, which is not ubiquitous and infects only pig cells. In addition to these viruses, recombinant PERV-A/C viruses have recently been described that (i) are able to infect human cells; (ii) are characterized by high titre replication; and (iii) are associated with proviruses that are de novo integrated in the DNA of somatic pig cells, but not yet in the pig germ line. The risks presented by PERV-A/C recombinant viruses could easily be eliminated by using pigs not containing PERV-C in their germ line, thereby effectively preventing recombination with PERV-A. Selection of PERV-C-free animals, if possible, therefore reduces the risk of PERV-A/C transmission to humans. Although it is unclear whether PERV-C may be transmitted in vivo from pig-to-pig, an infection of PERV-C-free animals with this virus may be prevented. To select pigs with low-level expression of PERV-A and PERV-B, it is recommended to apply assays based on real-time reverse transcriptase polymerase chain reaction (RT-PCR), which enables discrimination between pigs with high-level expression and low-level expression. Screening xenotransplant recipients for PERV transmission can be done in a number of ways. Provirus integration and PERV expression could theoretically be detected in peripheral blood mononuclear cells using PCR and RT-PCR. However, as the cells in which PERV replicates are still unknown, it is unclear whether this will be a reliable approach. Applying sufficiently sensitive assays to differentiate between transmission and chimerism is recommended. As can be commonly observed after retrovirus infection, the detection of virus-specific antibodies may indicate infection; however, the possibility of abortive infection, antigen exposure without infection, or cross-reactive response must be considered and explored as alternative explanations. On the other hand, it remains unclear whether the absence of specific antibodies indicates the absence of such infection, in particular if recipients of a xenotransplantation product are under chronic immunosuppression that could prevent antibody formation. Antibodies may be detected by Western blot or ELISA, using purified virus or recombinant viral proteins as antigens. Finally, it may be possible to detect cross-species PERV transmission by evaluating cells from the recipient for their in vitro potential of transmission to specified target cells (the human renal epithelial 293 cell line being the best example). There is no in vivo animal model for cross-species PERV transmission, and therefore it is not possible to validate monitoring assays for PERV transmission in an in vivo situation. Finally, virus safety of xenotransplantation is a fast-developing field, and new experimental findings will change existing strategies and introduce new ones.

An islet xenotransplantation product includes live cells from a non-human source, in this study, a pig source. A live product cannot be subjected to conventional disinfection, and therefore the source pig must be depleted of infectious agents that can transmit to the recipient and cause disease. Among other requirements, regulatory guidances specify that donor animals fulfill the designated pathogen-free (DPF) status. Donor pigs fulfilling DPF status are generally bred and maintained in biosecure facilities, where they are shielded from the outside by filtered air, disinfected water, and irradiated food that is certified free of any mammalian protein. All materials are autoclaved upon entry, and personnel enter by shower-in/shower-out, and are wearing special clothes. The operation of such facilities is in compliance with Current Good Manufacturing Practices. To ensure DPF status, pigs are brought into such facilities via Cesarean section, and the source pigs are kept as a closed herd. The DPF status cannot be realized for endogenous viruses, such as porcine endogenous retrovirus. Therefore, regulatory authorities require patient monitoring after xenotransplantation. Considering the infectious pathogen status and necessary regulatory compliance, it is recommended that organ procurement be conducted at the animal facility and that cell manufacturing facilities be located nearby. To enable assessment of as-yet unknown pathogens long after xenotransplantation, regulatory guidances mandate archiving donor materials for at least 50 yr. As this is essentially a public health issue, governmental institutions are urged to be responsible for the archive. Table of Contents • Introduction • Designated pathogen-free status • Biosecure barrier facility • Organ retrieval and islet manufacturing • Alternatives

BACKGROUND To avoid hyperacute rejection of xeno-organs, alpha1,3-galactosyltransferase knockout (GalT-KO) pigs have been produced. Galalpha1,3Gal determinant elimination may expose cryptic carbohydrate antigens and/or generate new antigens. This is the first biochemical study of carbohydrate antigens in GalT-KO pig organs. METHODS Neutral and acidic glycolipids were isolated from small intestine and pancreas of two GalT-KO and one wild-type (WT) pig. Glycolipid immune reactivity was tested on thin-layer chromatograms. Small intestine neutral glycolipids were separated by high-performance liquid chromatography and selected fractions were analyzed by proton nuclear magnetic resonance spectroscopy. Total gangliosides were quantified on thin-layer chromatograms and in microtiter wells. RESULTS Using Galalpha1,3nLc4 glycolipid reference, total Galalpha1,3Gal glycolipid antigens in the WT animal was estimated at about 30 microg (small intestine) and 3 microg (pancreas) per gram of dry tissue. Galalpha1,3Gal determinants were not detected in GalT-KO tissues at a detection limit of less than 0.25% (small intestine) and 0.5% (pancreas) of the WT tissues. Isoglobotriaosylceramide (iGb3) was absent but trace amounts of Fuc-iGb3 was found in both GalT-KO and WT pig small intestine. Blood group H type 2 core saccharide compounds were increased in GalT-KO pancreas. Total amount of gangliosides was decreased in GalT-KO tissues. The alpha1,3-galactosyltransferase acceptor, N-acetyllactosamine determinant, was not increased in GalT-KO tissues. Human serum antibodies reacted with WT organ Galalpha1,3Gal antigens and gangliosides, of which the ganglioside reactivity remained in GalT-KO tissues. CONCLUSIONS Knockout of porcine alpha1,3-galactosyltransferase gene results in elimination of Galalpha1,3Gal-terminated glycolipid compounds. GalT-KO genetic modification did not produce new compensatory glycolipid compounds reactive with human serum antibodies.

ObjectiveDuring ex vivo perfusion with human blood, homozygous galactosyl transferase knockout swine lungs exhibit prolonged survival (∼2 hours) relative to wild-type (500 mm Hg · min/L) and failed to support life within 21 minutes, two of three galactosyl transferase knockout lungs supported life, for 90 and 215 minutes, and displayed low peripheral vascular resistance (48 ± 12 mm Hg · min/L at 60 minutes), similar to the allogeneic control. Complement activation (delta C3a < 250 ng/mL through 60 minutes) and C5b-9 deposition were minimal in both galactosyl transferase knockout and membrane cofactor protein lungs. Neutrophils, monocytes, and platelets were rapidly sequestered in galactosyl transferase knockout and human membrane cofactor protein lung recipients, unlike the allogeneic control ( 0.5 nmol/L) was seen in the galactosyl transferase knockout recipients. Platelet activation (β-thromboglobulin rise > 200) and appearance of capillary congestion and vessel thrombosis confirmed coagulation activation associated with galactosyl transferase knockout lung failure.ConclusionsGalactosyl transferase knockout swine lungs are significantly protected in vivo from the physiologic consequences (increased pulmonary vascular resistance, capillary leak) associated with hyperacute lung rejection. As during ex vivo perfusion, dysregulated coagulation—thrombin elaboration, platelet activation, and intravascular thrombosis—mediates galactosyl transferase knockout lung xenograft injury.

Background: Hyperacute rejection (HAR) and early graft failure (EGF) have been described in a minority of pig-to-baboon heart transplants using organs transgenic for human complement regulatory proteins (hCRP). Here we investigate the role of coagulation cascade activation in the pathogenesis of HAR and EGF in a consecutive series where a high incidence of these outcomes was observed. Methods: Twenty-eight naive wild-caught Papio anubis baboons received heterotopic heart transplants from pigs transgenic for hDAF (n = 23) or hMCP (n = 5). Immunosuppression consisted of cyclosporine A, cyclophosphamide and MMF (n = 18) or anti-CD154 mAb (IDEC-131) and ATG (n = 10). Eleven received anti-Gal carbohydrates (GAS914, n = 8, or NEX1285, n = 3), of which four also underwent extracorporeal immunoadsorption (EIA), and 12 also received pharmacologic complement inhibitors (C1 INH, n = 9, or APT070, n = 3). Results: Excluding one technical failure, 14 of 27 transplants (11 hDAF, 3 hMCP) exhibited either HAR (n = 10) or EGF (n = 4). Surprisingly, neither complement inhibition (with C1 INH or APT070) nor anti-Gal antibody depletion with GAS914, NEX1285, or additional EIA consistently prevented HAR or EGF despite low or undetectable complement deposition. Strikingly, most grafts with HAR/EGF exhibited prominent fibrinogen and platelet deposition associated with systemic coagulation cascade activation, consistent with non-physiologic intravascular coagulation, in many instances despite little evidence for antibody-mediated complement activation. Conclusion: We conclude that dysregulated coagulation correlates closely with and probably causes primary failure of pig hearts transgenic for hCRP. These data support efforts to define effective strategies to prevent dysregulated coagulation in pig organ xenografts.

Xenotransplantation attracted interest from regulatory authorities, particularly after the demonstration of pig-to-human transmission of porcine endogenous retrovirus (1996). This added to the risk of a product, resulting in a Guidance of the US Food and Drug Administration (2003). This addresses the full flow chart in product manufacturing, starting with the designated pathogen-free status of the source animal; and special aspects regarding the recipient like informed consent and monitoring for infectious pathogens. Also archiving of records from the donor and recipient, as well as storage of samples is described. The European Medicines Agency issued a Guideline on xenogeneic cell therapy products (2009). Cell-based medicinal products are subject to specific regulations and directives, which apply also to xenogeneic products: the xenotransplant guidances/guidelines are an addition to these regulations. Noteworthy, acellular products like heart valves and decellularized cornea are not considered a cell therapy product, but rather a medical device with its own regulation. WHO issued relevant documents, especially about safety, and the International Xenotransplantation Association published consensus documents, a.o., addressing preclinical efficacy requirements before entering clinical trials. This manuscript presents an overview of the regulatory framework, with special focus on cell therapy products necause these are expected to reach the market first (i.e., pancreatic islets, hepatocytes and cellularized cornea); major illustrations are from the European situation. Albeit being complex, the regulation of xenotransplant products does not form a block in product development, but rather supports the introduction of efficacious and safe products to meet the medical need.

Background. The recent generation of α1,3-galactosyltransferase gene-knockout (GalT-KO) pigs has allowed investigation of the survival of GalT-KO pig organs in nonhuman primates. Methods. Heterotopic heart transplantation from GalT-KO pigs was carried out in baboons (n=8) using a human antihuman CD154 monoclonal antibody-based immunosuppressive regimen. Results. In six of the eight cases, graft survival extended to between approximately 2 and 6 months. All grafts developed thrombotic microangiopathy (TM). In particular, the clinical course of one baboon in which the graft functioned for 179 days is summarized. This baboon received aspirin (40 mg on alternate days) from day 4 in addition to heparin, which may have been a factor in the delay of onset and progression of TM and in prolonged graft survival. Maintenance therapy with anti-CD154 mAb, mycophenolate mofetil, and methylprednisolone was associated with persistently low numbers of CD3+CD4+ and CD3+CD8+ cells. Despite persisting depletion of these cells, no infectious complications occurred. Conclusions. It remains to be established whether TM is related to a very low level of natural preformed or T-cell-induced antibody deposition on the graft, inducing endothelial activation and injury, or to molecular incompatibilities in the coagulation mechanisms between pig and baboon, or to both. However, function of a pig organ in a baboon for a period approaching six months, which has not been reported previously, lends encouragement that the barriers to xenotransplantation will eventually be overcome. Copyright