The production of transgenic pigs for potential use in clinical xenotransplantation: microbiological evaluation

Debate over the infection hazards of pig-to-human xenotransplantation has focused mainly on the porcine endogenous retroviruses (PERV). However, hazards of exogenous infectious agents possibly associated with the xenograft have also been evaluated (Xenotransplantation 2000; 7: 143). We report the results of a health monitoring program demonstrating the exclusion of more than 80 potential pathogens from nine cohorts of pigs reared in a high welfare bioexclusion facility as potential xenograft source animals. A dynamic bacterial flora of pigs reared under barrier conditions was characterized, emphasizing the significance of monitoring for multiresistant antimicrobial sensitivity patterns. Evidence was found for exclusion of two commonly residual exogenous viruses, porcine cytomegalovirus and porcine lymphotropic herpesviruses, among a proportion of the cohorts tested. Finally, there was histopathological evidence for low grade pneumonitis among sentinel pigs, likely to have been associated with the use of quaternary ammonium disinfectants during the production process, indicating a need for review of toxicology data for disinfectant agents used in such bioexclusion systems. Intensive health monitoring programs, based upon regularly updated recommendations from the microbiological research community, will enable significant reductions in the potential hazards associated with pig-to-human xenotransplantation.     

The production of transgenic pigs for potential use in clinical xenotransplantation: baseline clinical pathology and organ size studies

This report describes the results of hematology, serum biochemistry, growth, and organ weight studies undertaken on pigs from nine cohorts of qualified pathogen free (QPF) pigs reared within a high welfare bioexclusion facility as potential organ source animals. Confirmation of the high health status of the pigs was given through total leukocyte counts and serum globulin concentrations that fell below the expected reference range for conventional pigs. The calculated mean growth rate for QPF pigs was found to exceed target rates set for optimum genotype commercial pig herds. Body weights of QPF pigs were compared with kidney, heart and liver weights at necropsy.     

The study of mitoxantrone as a potential immunosuppressor in transgenic pig renal xenotransplantation in baboons: comparison with cyclophosphamide

Mounting evidence suggests that delayed xenograft rejection (DXR) of discordant xenografts has a strong humoral component. To explore the possibility of targeting this humoral response more efficiently, we performed a preliminary study in baboons immunized against pig blood cells using the immunosuppressor mitoxantrone (Mx). The results from this study showed that, in comparison with cyclophosphamide (CyP), Mx induced a long-lasting depletion of circulating B cells within 6 days of its administration and delayed secondary anti-Gal antibody (Ab) responses to pig blood cell immunizations. Given these results, we next evaluated Mx in an in vivo model of pig to baboon renal xenotransplantation. We performed a series of renal xenotransplantations in baboons using human CD55-CD59 transgenic donor pigs. In the first group of baboons (Mx group; n = 4) Mx was administered 6 days prior to the day of transplantation, the objective being to perform the xenotransplantation in a context where the recipient would have few remaining circulating B cells and thus have an impaired capacity to mount an Ab response to the xenograft. We compared this group to a second group of baboons treated with CyP starting 1 day prior to transplantation (CyP group; n = 2). All baboons receiving Mx or CyP received an additional immunosuppression of cyclosporin A, mycophenolate mofetil and steroids. No hyperacute rejection was observed in either group but all xenografts underwent DXR. Mx did not show superiority to CyP in terms of graft survival with a mean survival time of 8 +/- 2 days compared with 9 days for both CyP-treated baboons. Neither CyP nor Mx decreased serum levels of pre-existing anti-Gal Abs but levels of these Abs decreased dramatically within 1 day of transplantation, likely reflecting their immediate trapping within the xenograft. Interestingly however, in contrast to CyP, Mx inhibited the return of anti-Gal immunoglobulin M (IgM) to the circulation, even at the time of rejection. Nevertheless, strong intragraft deposits of IgM, IgG and the activated complement complex C5b-9 were observed in biopsies at rejection. Furthermore, despite the expected profound depletion of circulating B cells by Mx within 6 days of its administration, biopsies from both groups at rejection displayed a mild B cell infiltrate accompanied by a strong macrophage and intermediate T-cell infiltration, the latter tending to be more abundant in Mx-treated animals. Our data show that in this particular model of pig to baboon xenotransplantation and at the dose used, Mx was not superior to CyP in conferring protection against rejection, despite its capacity to profoundly deplete circulating B cells and to inhibit anti-Gal Ab responses to xenografts. DXR was thus possible without the return of anti-Gal Abs and may have been mediated by the early fixation of pre-existing Abs with secondary complement activation. However, although Mx was not more efficient than CyP in controlling DXR, its capacity to deplete B cells and delay Ab recovery may be beneficial in the context of Gal knockout organ transplantation where the induced Ab response is likely to take precedence over the preformed response.     

New transgenic pigs for xenotransplantation,part 1

The hyperacute rejection response (HAR) after porcine‐to‐human xenotransplantation can now be reliably overcome. The next immunological hurdle is the acute vascular rejection (AVR) primarily caused by endothelial cell activation followed by disseminated intravascular coagulopathy, increased apoptosis and inflammatory symptoms. Several genes have been proposed to show protective effects against AVR, including human heme oxygenase‐I (hHO‐1) and human A20 (hA20) gene. HHO‐1 has primarily anti‐apoptotic and cell protective properties. The hA20 molecule possesses protective features against inflammatory and apoptotic stimuli in endothelial cells. Thus transgenic expression of these genes in pigs may be promising to prolong survival of porcine xenografts. We used somatic cell nuclear transfer (SCNT) for production of transgenic pigs. We produced pigs transgenic for human heme oxygenase 1 (hHO‐1) to evaluate the protective effects of that molecule and to compare it with other transgenes used to control of the hyperacute rejection response (HAR), e.g. the DAF transgenes which gave HAR protection in in vitro cell death assays. Importantly, hHO‐1 transgenic porcine aortic endothelial cells were significantly better protected against TNF‐α mediated apoptosis. In close collaboration with partners at the LMU Munich (Prof. Kupatt et al.) the transgenic pig lines were tested in an ischemia/reperfusion (I/R) circuit. After occlusion of the left anterior descending artery (LAD), hHO‐1 transgenic hearts had significantly smaller infarct lesions and concomitantly significantly better global myocardial function than size‐matched wild‐type controls. In close collaboration with partners at Hannover Medical University (Prof. Winkler et al.), hHO‐1 transgenic porcine kidneys were perfused with pooled human blood for the maximum period of 240 min without addition of C1‐Inhibitor in an ex vivo perfusion circuit. In parallel, we produced and characterized pigs that express hHO‐1 on a Gal^–/– background. Gal^–/–/hHO‐1 pig hearts were tested in the I/R circuit and preliminary results indicate a protective effect shown by decreased infarct size, less inflammation and improved global and regional myocardial function after LAD occlusion. Expression of hA20 from the CAGGS promoter was found in skeletal muscle, heart and PAECs. Cultured human A20‐transgenic PAECs showed significantly reduced apoptosis when compared to their wild type counterparts. Only partial protection of hA20‐transgenic pig hearts was observed after I/R. While infarct size was not different between the two groups after ischemic assault, hA20‐transgenic pig hearts showed a significantly better hemodynamic performance (determined as SES) than the wild type porcine hearts. MPO activity was reduced in transgenic vs. wild type hearts. We also produced pigs carrying shRNA constructs directed against PERV expression. These animals showed significantly reduced PERV‐expression for over 6 months compared to wild‐type and sham controls. This approach could improve the safety of porcine xenografts. We will now produce pigs carrying hHO‐1 on the Gal^–/–/hCD46 background. Tissues and organs from these animals will be tested in the previously established in vitro systems, and when positive results are obtained, hearts and kidneys will be transplanted into baboons. A second line of multi‐transgenic pigs will have both hA20 and shRNA against PERV expression on the Gal^–/–/hCD46/hHO‐1 background. The new somatic cloning protocol developed recently will allow rapid screening of promising transgene combinations and will ensure that we achieve our ambitious goals and move xenotransplantation closer to clinical application. This study was funded by grants from the Deutsche Forschungsgemeinschaft Ni 256/ 22‐1, ‐2, ‐3,‐4.     

Technique for heterotopic pig heart xenotransplantation in primates.

The primate is a commonly utilized model for the human immune response after heart transplantation. This report describes our experience with heterotopic abdominal transplantation of porcine hearts into primate recipients. Abdominal graft placement was surprisingly well tolerated, and we found this approach to be particularly useful in the setting of significant donor-recipient size mismatch. Continuous monitoring with an implantable monitoring system facilitated assessment of graft viability in awake recipients; progressive graft bradycardia and decreasing QRS amplitude were predictive of ensuing graft failure.     

Advanced transgenic strategies for modification of donor pigs in xenotransplantation

For xenotransplantation diverse rejection mechanisms are much more pronounced as compared to allotransplantation. The usage of genetically modified pigs, however, facilitates tailoring of donor animals for defined purposes (1). Although such genetic modifications were done decades ago, routine generation of transgenic pigs was not performed until somatic cell nuclear transfer (SCNT) was implemented for reproduction of large animals. SCNT avoids the production of mosaic founders and shifted the genetic modification towards the level of pig primary cells and, thus, improved the efficiency of transgenesis as it enables the generation of almost exclusively transgenic offspring, once the donor cells have been properly selected for vector integration (2). In addition, SCNT opened up the possibilities for any type of genetic modifications that has been developed for embryonic stem cells. In particular, the opportunity for site‐directed mutagenesis boosted the potential of genetically modified pig models. This was demonstrated for the removal of the α1,3‐galactosyl‐galactose epitopes by disruption of the GGTA1 gene which reduced the problem of hyperacute rejection to a minor topic in the xenotransplantation community. In the meanwhile more sophisticated methods such as modified bacterial artificial chromosomes, viral vectors or site‐specific nucleases further increased the potential for site‐directed mutagenesis in pig (3, 4). The latter technology is based on the introduction of a DNA double strand break by a nuclease that is directed to the target site by specific DNA‐binding domains. Mutations are introduced by erroneous repair through non‐homologous end joining. Alternatively, a targeting vector can be used in combination with a site‐specific nuclease to introduce a targeted modification via homologous recombination. Other advanced transgenic strategies such as the two‐vector based TetOn technology for inducible transgene expression are routinely performed in the mouse, but the significantly longer generation time of large animals hampered its straight translation into the pig. As we demonstrated recently, sequential transgenesis by repeated rounds of SCNT is a practicable way to evaluate biological transgene function in founder animals within a considerable time frame (5). In addition to technological improvements at the cellular as well as at the embryonic level, the recent boost of genomic information from multiple species and its bioinformatics analysis improved the design of transgenic pigs. As for many problems cell type‐specific expression of a transgene is desired, the definition of appropriate regulatory elements is required. Many of those have been described in the mouse, but in general endogenous sequences are seen as superior to the usage of murine promoters in the pig. Multiple‐sequence alignments from diverse mammalian species facilitate the identification of the orthologous region of murine regulatory elements in the pig. Interestingly, with the increasing number of transgenes available for xenotransplantation approaches, the breeding aspect gained new attention. It is clear that for optimized donor pigs multiple transgenes should be combined and, on the long run, mendelian transgene segregation should be avoided by using novel transgene approaches. However, until such “all‐in‐one” vectors are available, the most straightforward strategy is the combination of existing and properly characterized lines by conventional breeding strategies. These require profound organization and logistics to resolve the conflicting aspects of transgene segregation and inbreeding and to enable the systematic evaluation of donor herds for microbial contamination. Thus, the task field of donor pig suppliers in xenotransplantation expanded from relatively simple reproductive stints to advanced design and construction of novel transgenic pigs and organizing challenges regarding continuous supply of donor animals. References 1. Klymiuk N, Aigner B, Brem, et al. Genetic modification of pigs as organ donors for xenotransplantation. Mol Reprod Dev 2010; 77: 209. 2. Aigner Bet al. Transgenic pigs as models for translational biomedical research. J Mol Med (Berl) 2010; 88, 653. 3. Klymiuk Net al. Sequential targeting of CFTR by BAC vectors generates a novel pig model of cystic fibrosis. J Mol Med (Berl) 2011. 4. Hauschild Jet al. Efficient generation of a biallelic knockout in pigs using zinc‐finger nucleases. Proceedings of the National Academy of Sciences of the United States of America 2011; 108: 12013. 5. Klymiuk Net al. First inducible transgene expression in porcine large animal models. FASEB J 2011.     

Establishment of genetically modified pig animal models for xenotransplantation

Due to the shortage of available human donor organs, xenotransplantation is an alternative to allotransplanation. Pig is the favoured donor animal because of its similarities to man in size, anatomy and physiology as well as ethical reasons and questions of infectivity. On the other hand the genetic distance between human and porcine species requires comprehensive precautions to overcome immunological hurdles. Genetic alteration of the porcine genome is preferred to diminish rejection mechanisms rather than exposing human recipients to severe immunological treatment. Since the invention of somatic cloning, nuclear transfer is the most prominent way to establish transgenic large animals. However, the generation of a transgenic animal model is still a bold venture in costs of time and money. We have established somatic cloning of pigs and the generation of transgenic fibroblasts at our institute for transgenic pig production in a feasible extent.     

Genetic and functional evaluation of the level of inbreeding of the Westran pig: a herd with potential for use in xenotransplantation

BACKGROUND: The Westran pig has been purposely inbred for use in xenotransplantation. The herd originated in the wild from a limited gene pool and has been inbred by repeated full-sib matings for nine generations. METHODS: The aim of this study was to evaluate the level of inbreeding by functional assays, such as bi-directional MLR and reciprocal skin grafts between herd members, and by genetic analysis using highly polymorphic genetic markers to calculate the level of inbreeding. RESULTS: The MLR between herd members were non-reactive whereas there was a prompt response to third party pig lymphocytes, indicative of a normal immune responsiveness in Westran pigs but isogenicity of the major histocompatibility complex. Skin grafts between male siblings or female sibling skin grafts on male recipients showed prolonged survival but with few exceptions did not survive beyond 100 days suggesting that by the fifth generation the Westran herd was still mismatched at minor histocompatibility antigens. This level of functional inbreeding was confirmed by microsatellite analysis of highly polymorphic markers, which showed that 52 of 53 chromosomally dispersed markers were fixed by the ninth generation. This level of fixation was consistent with 19 to 20 generations of full-sibling inbreeding. The calculated inbreeding coefficient at generation 10 was 0.98159. CONCLUSIONS: This analysis confirms that the Westran pig is highly inbred and we propose that analysis of chromosomally dispersed highly polymorphic markers is an accurate and reproducible method for assessing the level of inbreeding of a pig herd.     

Safe use of anti‐CD154 monoclonal antibody in pig islet xenotransplantation in monkeys

Anti‐CD154mAb is a powerful co‐stimulation blockade agent that is efficacious in preventing rejection, even in xenogeneic settings. It has been used in the majority of successful long‐term pig‐to‐non‐human primate islet transplantation models. However, its clinical use was halted as a result of thromboembolic complications that were also observed in preclinical and clinical organ transplantation models. An anti‐CD154mAb was administered to 14 streptozotocin‐induced diabetic cynomolgus monkey recipients of porcine islets, some of which received the agent for many months. Monkeys were monitored for complications, and blood monitoring was carried out frequently. After euthanasia, multiple biopsies of all organs were examined for histological features of thromboembolism. Anti‐CD154mAb prevented rejection of genetically engineered pig islets in all monkeys. No significant complications were attributable specifically to anti‐CD154mAb. There was no evidence of thromboembolism in multiple histological sections from all major organs, including the brain. Our data suggest that in diabetic monkeys with pig islet grafts, anti‐CD154mAb would appear to be an effective and safe therapy, and is not associated with thromboembolic complications.