Multi‐transgenic pigs for xenoislet transplantation

Background: Islets obtained from genetically‐engineered (GE) pigs with Gal‐knockout (GTKO) and high expression of hCD46 have shown long‐term function and successful correction of insulin independence in nonhuman primates (NHP). Two anti‐coagulant genes (hTFPI and hCD39), and an immunosuppressive gene (pCTLA4Ig) have been added to the GTKO/hCD46 background, using an islet‐specific expression system, for inhibition of thrombosis, inflammation, early islet loss, and rejection. Transplant outcomes are being tested in a diabetic monkey model using adult islets from these multi‐transgenic pigs. Methods: Three individual vectors (CD39^ins, TFPI^ins and CTLA4Ig^ins), each under control of the rat insulin II promoter and mouse PDX‐1 enhancer, were transfected alone or in combination into GTKO/hCD46 pig fibroblast cells. GTKO/hCD46/CD39^ins, GTKO/hCD46/TFPI^ins/CTLA4Ig^ins, and GTKO/hCD46/CD39^ins/TFPI^ins/CTLA4Ig^ins cell lines were used for nuclear transfer (NT) to produce pregnancies. Cloned pigs were analyzed for transgene expression in pancreas, heart, and liver by IHC, Western, and real‐time PCR. Glucose metabolism in the various GE pigs was determined by measurement of blood glucose, c‐peptide, and via intravenous glucose tolerance tests (ivGTT) or arginine stimulation test. GE islets from adult pigs were transplanted intraportally into STZ‐diabetic monkeys using an ATG/MMF/anti‐CD154 immunosuppression regimen. Results: Viable multi‐transgenic pigs with three, four, or five different genetic modifications were produced. Three different pig lines were established: (i) GTKO/hCD46/CD39^ins, (ii) GTKO/hCD46/TFPI^ins/CTLA4Ig^ins, and (iii) GTKO/hCD46/CD39^ins/TFPI^ins/CTLA4Ig^ins. IHC revealed robust islet‐specific expression of CD39, TFPI and CTLA4Ig in pancreas, with only background expression in heart and liver. Western analysis showed strong pCTLA4Ig expression only in the pancreas. Blood glucose metabolism was normal in all GE pigs tested. Islets from GE pigs containing the TFPI transgene, when mixed with human blood, showed prolonged clotting times. Early islet loss (IBMIR) was reduced 5–7 fold in NHP transplants using islets from 4‐GE and 5‐GE pigs vs. 2‐GE pig islets. Conclusions: (i) Healthy multi‐transgenic pigs with islet‐specific expression of hCD39, hTFPI, and pCTLA4Ig were produced. (ii) Glucose metabolism was normal in these GE pigs. (iii) GE islets showed anti‐coagulation activity in vitro, and protection from IBMIR in vivo in NHP transplants. (iv) GE islets transplanted into diabetic monkeys demonstrated prolonged survival, function, and complete normalization of blood glucose levels for up to 1 year.     

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.     

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.     

Generation of HLA-DP transgenic pigs for the study of xenotransplantation.

The shortage of human organs has prompted scientists to seek xenogeneic sources of donors. To date, DAF, MCP, and CD59 transgenic pigs have been generated to inhibit hyperacute rejection. However, besides hyperacute rejection, acute and chronic rejection must also be considered in the use of porcine organs for xenotransplantation. The role of HLA-II in transgenic xeno-organ transplantation remains to be elucidated. By microinjecting 1655 embryos, we have generated one stillborn HLA-DR and two live HLA-DP transgenic pigs: P113-7 (male, carrying one copy of exogene) and P113-8 (female, carrying 2-3 copies of exogenes). The gene status of the live transgenic pigs was confirmed by PCR, Southern blot, and PCR product sequencing analysis. The expression of transgenes in these transgenic pigs were confirmed by RT-PCR analysis and immunohistochemical staining of frozen sections of ear tissue.     

Engineering pigs for xenotransplantation

Xenotransplantation using tissues and organs from genetically modified pigs has the potential to fulfil the serious shortage of material available for human transplantation. However, considerable immunological barriers must be overcome before such discordant grafts can be used. The generation of α(1,3)‐galactosyltransferase knock out pig lines has surmounted the initial obstacle of hyperacute rejection. Further improvements are now necessary to provide adequate protection against acute humoral xenograft rejection. The expression of human complement regulator transgenes in existing transgenic animals is inadequate, and a means of achieving abundant uniform expression in the organ is required. Recent findings have also highlighted the need for additional transgenes to alleviate incompatibilities between the porcine and human anticoagulation and anti‐inflammatory systems. Generating multi‐transgenic animals by breeding individual transgenic animals is inefficient and incurs several problems, including: independent Mendelian segregation of multiple transgenes; failure of different transgenes to co‐express at the right time or in the correct tissue; and an increased risk of insertional mutagenesis due to multiple integration events. A novel approach is therefore required to produce what are likely to be increasingly complex multi‐transgenic animals. Ideally, all transgenes should reside at a single Mendelian locus. They should not be subject to the position effect. There should be no limitation on transgene size to allow inclusion of substantial regulatory regions, or genes as multiple copies to obtain abundant expression. Finally, insertional mutagenesis should be excluded if possible. One possible way to achieve these is to use artificial chromosome vectors. De novo formed human artificial chromosomes are being developed as vectors for human gene therapy. The feasibility of this and alternative approaches to deliver sets of xenoprotective transgenes into pigs is being explored. This work is being carried out in close collaboration with the Xeno‐Forschergruppe (FOR 535) and is supported through funding provided by the DFG.     

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.     

Possible risks posed by single‐stranded DNA viruses of pigs associated with xenotransplantation

Routine large‐scale xenotransplantation from pigs to humans is getting closer to clinical reality owing to several state‐of‐the‐art technologies, especially the ability to rapidly engineer genetically defined pigs. However, using pig organs in humans poses risks including unwanted cross‐species transfer of viruses and adaption of these pig viruses to the human organ recipient. Recent developments in the field of virology, including the advent of metagenomic techniques to characterize entire viromes, have led to the identification of a plethora of viruses in many niches. Single‐stranded DNA (ssDNA) viruses are the largest group prevalent in virome studies in mammals. Specifically, the ssDNA viral genomes are characterized by a high rate of nucleotide substitution, which confers a proclivity to adapt to new hosts and cross‐species barriers. Pig‐associated ssDNA viruses include torque teno sus viruses (TTSuV) in the Anelloviridae family, porcine parvoviruses (PPV), and porcine bocaviruses (PBoV) both in the family of Parvoviridae, and porcine circoviruses (PCV) in the Circoviridae family, some of which have been confirmed to be pathogenic to pigs. The risks of these viruses for the human recipient during xenotransplantation procedures are relatively unknown. Based on the scant knowledge available on the prevalence, predilection, and pathogenicity of pig‐associated ssDNA viruses, careful screening and monitoring are required. In the case of positive identification, risk assessments and strategies to eliminate these viruses in xenotransplantation pig stock may be needed.     

Genetic modification of pigs for solid organ xenotransplantation

Xenotransplantation of solid organs will only ever become a clinical reality with genetic modification of the pig, which is now widely accepted as the most likely donor species for humans. The understanding of the barriers to xenotransplantation has required advances in genetic technologies to resolve these problems. Hyperacute rejection has been overcome by overexpression of complement regulatory proteins or targeted disruption of the enzyme associated with the major carbohydrate xenoantigen. The subsequent barriers of disordered coagulation, induced antibody, and cell-mediated rejection remain challenging. The mechanisms for these incompatibilities are being deciphered, and multiple genetic manipulations to resolve these issues are currently in progress. Moreover, new technologies offer help to producing sizeable numbers of modified pigs in a timely manner. This article retraces the basis and foreshadows progress of the genetically modified pig for xenotransplantation as it advances toward the clinic.     

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.     

Characterization of human CD55 and CD59 transgenic pigs and kidney xenotransplantation in the pig-to-baboon combination

New transgenic pigs expressing combinations of regulators of complement activation and other molecules are needed to resist xenograft hyperacute rejection (HAR) and to further analyze and treat xenograft rejection. Double transgenic pigs for human CD55 (hCD55) and human CD59 (hCD59) using the promoter of the human elongation factor 1 alpha gene were generated, and their kidneys were transplanted into nonimmunosuppressed baboons. hCD55 and hCD59 were mainly expressed by the endothelial cells, and these cells showed increased resistance to complement-mediated lysis. Baboons receiving kidneys from hCD55hCD59 pigs survived for 5 and 6 days, and displayed alterations in coagulation. Thrombocytopenia and platelet microthrombi were present within the kidneys. Nontransgenic kidneys showed HAR in less than 2 days. Kidneys from pigs expressing hCD55hCD59 displayed protection against HAR in the absence of immunosuppression. Rejection was associated with coagulopathy leukocyte infiltration and a rebound of anti-alpha Gal antibodies.     

Systematic design,production and breeding of multi‐transgenic donor pigs

The continuously increasing shortage of donor organs for allotransplantation demands the development of alternative treatments. The implementation of xenotransplantation suffers from various pathways of rejection and incompatibilities between recipient and donor tissues. Usage of transgenic donor pigs might overcome these problems, but the generation of transgenic animals by nuclear transfer is costly and teadious, the combination of several transgenes represents considerable efforts and the permanent production of donor pigs requires optimization between the conflicting issues of transgene segregation and high inbreeding coefficient. The establishment of novel transgenes is normally biased, which means that with increasing knowledge on the diverse mechanisms of xenograft failure, novel transgene variants arise. In addition, the poor knowledge about the porcine genome and site‐specific regulation in pigs requires concise planning and pre‐evaluation of bio‐informatic data. The generation of transgenic pig follows the conventional and well established strategies in mouse, with the exception that no embryonic stem cells are available in the pig. But the establishment of transgenic primary cells, their usage as donors for somatic cell nuclear transfer and the transfer of cloned embryos into synchronized gilts represent a reasonable alternative, conventionally resulting in suitable founder animals within a few litters. The characterization of the founders depends on the site of expression and, thus, influences the strategy of founder reproduction. The lateron combination with other transgenes is challenging and normally requires implementation of the transgene into an existing breeding herd. Thus, the breeding concept has to be designed to (i) to establish a core herd, (ii) to allow the addition of novel transgenes to the herd and (iii) to support the maintainence of the breeding herd by continuous re‐juvenation. The latter is of certain importance, as transgenes normally segregate according to the Mendelian pattern of inheritance. This aspect would favour a homozygous transgene status for all breeding animals. However, such a strategy will result in dramatically increasing inbreeding coefficients and, consequently, reduce fertility and litter sizes. Taken together, both, the permanent production of multi‐transgenic donor animals as well as the development of novel transgenes and their integration into a breeding herd is a logistic challenge that requires long‐term planning but also flexibility regarding the transgene combination which depend is governed by the increasing insight in the diverse mechanisms of xenograft failure.