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Background: The present study reports the development of a sensitive dot blot protocol for determining the level of preformed antibodies against porcine heart valve tissue derived from wild‐type (WT) and α‐Gal‐KO (GGTA1‐KO) pigs in human sera. Methods: The assay uses decellularized and solubilized heart valve tissue; antibody binding found in this dot blot assay could be correlated with antibody titers of preformed anti‐α‐Gal and anti‐Neu5Gc antibodies detected by a sensitive ELISA. Results: The ultimate protocol had an inter‐assay variance of 9.5% and an intra‐assay variance of 9.2%, showing that the test is reliable and highly reproducible. With the aid of this dot blot assay, we found significant variation with regard to antibody contents among twelve human sera. Binding of preformed antibodies to WT tissue was significantly higher than to GGTA1‐KO tissue. Conclusions: The dot blot assay described herein could be a valuable tool to measure preformed antibody levels in human sera against unknown epitopes on decellularized tissue prior to implantation. Ultimately, this prescreening may allow a matching of the porcine xenograft with the respective human recipients in demand and thus may become an important tool for graft long‐term survival similar to current allotransplantation settings. [ABSTRACT FROM AUTHOR]

Xenotransplantation of pancreatic islets offers a promising alternative to overcome the shortage of allogeneic donors. Despite significant advances, either immune rejection or oxygen supply in immune protected encapsulated islets remains major bottlenecks for clinical application. To decrease xenogeneic immune responses, we generated tissue engineered swine leucocyte antigen (SLA)‐silenced islet cell clusters (ICC). Single‐cell suspensions from pancreatic islets were generated by enzymatic digestion of porcine ICCs. Cells were silenced for SLA class I and class II by lentiviral vectors encoding for short hairpin RNAs targeting beta2‐microglobulin or class II transactivator, respectively. SLA‐silenced ICCs‐derived cells were then used to form new ICCs in stirred bioreactors in the presence of collagen VI. SLA class I silencing was designed to reach a level of up to 89% and class II by up to 81% on ICCs‐derived cells. Xenogeneic T cell immune responses, NK cell and antibody‐mediated cellular‐dependent immune responses were significantly decreased in SLA‐silenced cells. In stirred bioreactors, tissue engineered islets showed the typical 3D structure and insulin production. These data show the feasibility to generate low immunogenic porcine ICCs after single‐cell engineering and post‐transduction islet reassembling that might serve as an alternative to allogeneic pancreatic islet cell transplantation. [ABSTRACT FROM AUTHOR]

Background Xenotransplantation is considered to be a promising solution to the growing demand for suitable donor organs for transplantation. Despite tremendous progress in the generation of pigs with multiple genetic modifications thought to be necessary to overcoming the severe rejection responses after pig-to-non-human primate xenotransplantation, the production of knockout pigs by somatic cell nuclear transfer ( SCNT) is still an inefficient process. Producing genetically modified pigs by intracytoplasmic microinjection of porcine zygotes is an alluring alternative. The porcine GGTA1 gene encodes for the α1,3-galactosyltransferase that synthesizes the Gal epitopes on porcine cells which constitute the major antigen in a xenotransplantation setting. GGTA1- KO pigs have successfully been produced by transfecting somatic cells with zinc-finger nucleases ( ZFNs), transcription activator-like effector nucleases ( TALENs), or CRISPR/Cas targeting GGTA1, followed by SCNT. Methods Here, we microinjected a CRISPR/Cas9 vector coding for a single-guide RNA (sg RNA) targeting exon 8 of the GGTA1 gene into the cytoplasm of 97 in vivo-derived porcine zygotes and transferred 86 of the microinjected embryos into three hormonally synchronized recipients. Fetuses and piglets were analyzed by flow cytometry for remaining Gal epitopes. DNA was sequenced to detect mutations at the GGTA1 locus. Results Two of the recipients remained pregnant as determined by ultrasound scanning on day 25 of gestation. One pregnancy was terminated on day 26, and six healthy fetuses were recovered. The second pregnancy was allowed to go to term and resulted in the birth of six healthy piglets. Flow cytometry analysis revealed the absence of Gal epitopes in four of six fetuses (66%), indicating a biallelic KO of GGTA1. Additionally, three of the six live-born piglets (50%) did not express Gal epitopes on their cell surface. Two fetuses and two piglets showed a mosaicism with a mixed population of Gal-free and Gal-expressing cells. Only a single piglet did not have any genomic modifications. Genomic sequencing revealed indel formation at the GGTA1 locus ranging from +17 bp to −20 bp. Conclusions These results demonstrate the efficacy of CRISPR/Cas to generate genetic modifications in pigs by simplified technology, such as intracytoplasmic microinjection into zygotes, which would significantly facilitate the production of genetically modified pigs suitable for xenotransplantation. Importantly, this simplified injection protocol avoids the penetration of the vulnerable pronuclear membrane, and is thus compatible with higher survival rates of microinjected embryos, which in turn facilitates production of genetically modified piglets. [ABSTRACT FROM AUTHOR]

Background Xenogeneic thrombotic microangiopathy ( TMA) and acute vascular rejection ( AVR) prevent long-term survival of porcine xenografts after transplantation into non-human primates. Preformed xenoreactive natural antibodies ( XNA) cause endothelial damage and activate the complement system. Mechanisms of xenogeneic coagulation and platelet activation are only poorly characterized. Methods A microfluidic flow chamber was used to study platelet activation and thrombus formation of human platelet-rich plasma ( PRP) upon perfusion over wild-type ( WT) or α-1,3- galactosyltransferase knockout ( GTKO) and human CD46 ( hCD46) transgenic porcine aortic endothelial cells ( PAEC). Activation of plasma coagulation (thrombin-anti-thrombin complex; TAT) and complement (C3a, C5a) was studied in human platelet-free plasma ( PFP) after co-incubation with PAEC. The activation of PAEC (E-Selectin, tissue factor, ICAM-1, ICAM-2, VCAM-1) was studied after incubation with human serum. Eculizumab (200 μg/ml) was used to inhibit terminal complement activation in all experiments. Results WT- PAEC perfused with human PRP showed thrombus formation at different shear rates (3 dyn/cm2: 23 ± 10%; 10 dyn/cm2: 17 ± 10% of flow chamber viewing field). GTKO/ hCD46 PAEC exhibited reduced, but not fully prevented thrombus formation (3 dyn/cm2: 12 ± 12%). Porcine PRP caused little or no thrombus formation (3.0 ± 4% and 0.5 ± 0.9%, respectively). Flow cytometry of human platelets after perfusion over WT- PAEC revealed an increase in platelet CD62P expression (29.5 ± 3%), compared to non-perfused PRP (7 ± 2%) or PRP running through empty flow chambers (12.7 ± 0.3%). Incubation of human PFP with PAEC resulted in an increase of TAT that correlated with C5a activation. Specific inhibition of complement by eculizumab prevented thrombus formation ( WT- PAEC: 1.6 ± 2% at 3 dyn/cm2 and 0.24 ± 0.33% at 10 dyn/cm2, GTKO/ hCD46 PAEC: 0.2 ± 0.3% at 3 dyn/cm2) as well as activation of coagulation and platelets. Induction of endothelial E-Selectin and VCAM-1 in WT- PAEC upon incubation with human serum was significantly reduced by eculizumab. Eculizumab did not reduce thrombin generation capacity of human PRP or normal platelet aggregation. Conclusion Thrombus formation in this ex vivo model of xenogeneic TMA was closely linked with complement activation. Specific inhibition of complement C5 by eculizumab prevented endothelial cell activation, but also coagulation and platelet activation without compromising thrombin generation capacity of human blood or normal platelet function. [ABSTRACT FROM AUTHOR]

The heterotopic thoracic pig-to-baboon heart transplantation has been established by our group as a safe preclinical model. Since the recipient's own heart remains in place, it is possible to evaluate immunological reactions of various types and the symptoms of the thrombotic microangiopathy under working heart conditions. However, these experiments are complex and costly. In order to limit the number of transplants, we tested our multi-transgenic porcine donor hearts in an ex-vivo perfusion model first. Ex-vivo model: Both beating ventricles were perfused with heparinized freshly drawn human whole blood using a centrifugal pump and a membrane oxygenator. During 3 h of observation, cardiac function parameters were obtained continuously; specimens from the perfusate, coronary sinus blood, and myocardial biopsies were assessed. Various genetically modified porcine donor hearts were tested; we are currently evaluating the impact of human thrombomodulin on the porcine microcirculation; the efficacy of complement regulation is another work package. Pig-to-baboon heterotopic cardiac xenotransplantation: In our latest group, seven baboons received five double (Gal-KO/hCD46), and two triple (Gal-KO/hCD46/hTM resp./HLA-E) transgenic pig hearts. Our immunosuppressive regimen included preoperative anti-CD20-antibody, bortezomib, dexamethasone and cyclophosphamide; postoperatively, ATG, tacrolimus, MMF, anti-CD20-antibody, bortezomib and dexamethasone were administered. Total lymph node irradiation (6 Gy) was applied on postoperative day five. The triple transgenic hearts survived 35 and 37 days respectively. Both hearts maintained function throughout the experiment. Both recipients succumbed to fungal infections. Humoral rejection was seen only once. The next barrier appears to be the complex event of thrombotic microangiopathy that needs to be addressed with the additional expression of human thrombomodulin strategies. [ABSTRACT FROM AUTHOR]

Semaan M, Kaulitz D, Petersen B, Niemann H, Denner J. Long-term effects of PERV-specific RNA interference in transgenic pigs. Xenotransplantation 2012; 19: 112-121. © 2012 John Wiley & Sons A/S. Abstract: Background: Porcine endogenous retroviruses (PERVs) represent a risk of xenotransplantation using porcine cells, tissues, or organs, as they are integrated in the porcine genome and have been shown to be able to infect human cells in vitro. To increase viral safety by RNA interference, transgenic pigs expressing a PERV-specific small hairpin (sh)RNA targeted to a highly conserved sequence in the pol gene (pol2) were generated in which expression of PERVs was reduced (Xenotransplantation, 15, 2008, 38). However, it remains to be shown how long expression of the shRNA and the RNA interference is effective in reducing PERV expression. Methods: To analyze the long-term duration of RNA interference, expression of the PERV-specific pol2 shRNA and inhibition of PERV expression was studied repeatedly in fibroblasts and peripheral blood mononuclear cells (PBMCs) of transgenic pigs over a period of 3 yr, when animals were sacrificed and expression was studied in different organs. Expression of the PERV-specific shRNA was measured using a newly developed real-time PCR, and expression of PERV was measured using a PERV-specific real-time PCR. Results: Over a period of 3 yr, PERV-specific shRNA and green fluorescent protein (GFP) as reporter of the vector system were consistently expressed in transgenic animals. PERV expression was significantly reduced during the entire period. Levels of PERV and shRNA expression were different in the various organs. PERV expression was highest in the spleen and the lungs and lowest in liver and heart. However, in all organs of the transgenic pigs, PERV expression was inhibited compared with the vector control animals. Conclusions: Transgenic pigs expressing PERV-specific shRNA maintained their specific RNA interference long term, suggesting that PERV expression in the xenotransplants will be suppressed over extended periods of time. [ABSTRACT FROM AUTHOR]

Zinc-finger nucleases (ZFNs) are powerful tools for producing gene knockouts (KOs) with high efficiency. Whereas ZFN-mediated gene disruption has been demonstrated in laboratory animals such as mice, rats, and fruit flies, ZFNs have not been used to disrupt an endogenous gene in any large domestic species. Here we used ZFNs to induce a biallelic knockout of the porcine α1,3-galactosyltransferase (GGTA1) gene. Primary porcine fibroblasts were treated with ZFNs designed against the region coding for the catalytic core of GGTA1, resulting in biallelic knockout of ∼1% of ZFN-treated cells. A galactose (Gal) epitope counter-selected population of these cells was used in somatic cell nuclear transfer (SCNT). Of the resulting six fetuses, all completely lacked Gal epitopes and were phenotypically indistinguishable from the starting donor cell population, illustrating that ZFN-mediated genetic modification did not interfere with the cloning process. Neither off-target cleavage events nor integration of the ZFN-coding plasmid was detected. The GGTA1-KO phenotype was confirmed by a complement lysis assay that demonstrated protection of GGTA1-KO fibroblasts relative to wild-type cells. Cells from GGTA1-KO fetuses and pooled, transfected cells were used to produce live offspring via SCNT. This study reports the production of cloned pigs carrying a biallelic ZFN-induced knockout of an endogenous gene. These findings open a unique avenue toward the creation of gene KO pigs, which could benefit both agriculture and biomedicine. [ABSTRACT FROM AUTHOR]

Oropeza M, Petersen B, Carnwath JW, Lucas-Hahn A, Lemme E, Hassel P, Herrmann D, Barg-Kues B, Holler S, Queisser A-L, Schwinzer R, Hinkel R, Kupatt C, Niemann H. Transgenic expression of the human A20 gene in cloned pigs provides protection against apoptotic and inflammatory stimuli. Xenotransplantation 2009; 16: 522–534. © 2009 John Wiley & Sons A/S. Porcine organs with transgenic expression of anti-apoptotic and anti-inflammatory genes like the human A20 gene (hA20), a tumor necrosis factor-alpha (TNF-α)-inducible gene, may control the acute vascular rejection (AVR) of porcine xenografts. The human A20 molecule possesses protective features against inflammatory and apoptotic stimuli in various cell types including endothelial cells, rendering it a promising candidate for transgenic pig production in the context of xenotransplantation. Here, we produced pigs transgenic for hA20 and investigated whether hA20-transgenic porcine aortic endothelial cells (PAECs) were resistant against the induction of apoptosis in vitro and to what extent hA20-transgenic porcine hearts were protected against ischemia/reperfusion (I/R) injury. Methods: Porcine fetal fibroblasts (PFFs) were transfected with the vector pCAGGSEhA20-IRESNEO containing a chicken β-actin/rabbit β-globin (CAGGS)-promoter element, known to provide ubiquitous gene expression in both mice and pigs. Transfected PFFs were then used in somatic cell nuclear transfer (SCNT). Three hA20-transgenic pigs were killed for PAEC isolation and organ mRNA and protein expression analysis by reverse transcriptase-polymerase chain reaction (RT-PCR), Northern and Western Blotting. PAECs were tested for susceptibility to apoptosis after TNF-α challenging and triggering of the CD95(Fas)/CD95Ligand pathway. Five transgenic and three wild type animals were subjected to an I/R experiment followed by measurement of infarct size, myeloperoxidase (MPO) activity and subendocardial segmental shortening (SES) to assess protective effects of hA20 in the porcine myocardium. Results: The hA20-transgenic pigs developed normally and expression of hA20 was found in skeletal muscle, heart and PAECs. Cultured human A20-transgenic PAECs showed significantly reduced apoptosis when compared to their wild type counterparts and were less susceptible to the induction of cell death by CD95(Fas)L. Only partial protection of hA20-transgenic pig hearts was observed after I/R. While infarct size did not differ between the two groups after ischemic assault, hA20-transgenic porcine hearts showed significantly lower MPO activity and better hemodynamic performance (determined as SES) than their wild type counterparts. Conclusions: The hA20 gene was for the first time functionally expressed in transgenic pigs. Although the CAGGS is a ubiquitous promoter element, expression was restricted to heart, skeletal muscle and PAECs of transgenic animals. Cultivated hA20-transgenic PAECs were protected against TNF-α-mediated apoptosis, and partially protected against CD95(Fas)L-mediated cell death; cardiomyocytes were partially protected in I/R. These findings reveal hA20 as a promising molecule for controlling AVR in multi-transgenic pigs for xenotransplantation studies. [ABSTRACT FROM AUTHOR]

Petersen B, Ramackers W, Tiede A, Lucas-Hahn A, Herrmann D, Barg-Kues B, Schuettler W, Friedrich L, Schwinzer R, Winkler M, Niemann H. Pigs transgenic for human thrombomodulin have elevated production of activated protein C. Xenotransplantation 2009; 16: 486–495. © 2009 John Wiley & Sons A/S. The inability of porcine thrombomodulin (TM) to activate human anticoagulant protein C after pig-to-human xenotransplantation may lead to an aberrant activation of coagulation with microthrombosis and ultimately failure of the transplanted organ. Here, we describe the production of triple-transgenic pigs expressing hCD59/DAF and human thrombomodulin (hTM) and tested hTM-transgenic fibroblasts obtained from these pigs for their ability to activate human protein C in a new in vitro assay. Methods: Fibroblast cell cultures were established from a hCD59/DAF transgenic pig and transfected with a vector coding for hTM under transcriptional control of the CMV promoter. Transfected cells were analyzed for integration and expression of the hTM vector by PCR and RT-PCR. One cell clone was used as donor for somatic cell nuclear transfer to produce triple transgenic (CD59/DAF/hTM) pigs. Pigs were characterized in detail with regard to hTM integration and expression by PCR, RT-PCR, Northern blot, Western blot, immunostaining, and FACS analysis. Fibroblasts from hTM-transgenic pigs were analyzed in a new in vitro hTM coactivity assay to assess the production of activated protein C (aPC) and results were compared to those from wild-type controls. Results: In total, 1040 cloned transgenic embryos were transferred to eight recipients. Five recipients remained pregnant and delivered 22 piglets. Expression of hTM was detected in all xenorelavant organs including heart, liver, kidney, lung, and pancreas. The lowest levels of expression were found in lung and liver while all animals showed a strong, but frequently patchy expression pattern of hTM in heart, kidney, and pancreas. The hTM cofactor activity (ranging on a scale from 5–18 U/105 cells/2h) was significantly higher in fibroblasts of hTM-transgenic clones compared to wild-type porcine fibroblasts (1.7 U/105 cells/2h). Conclusions: For the first time, healthy hTM-transgenic pigs could be successfully generated by somatic cell nuclear transfer. hTM can be expressed in porcine organs without perturbation of the porcine coagulation system. hTM-transgenic porcine fibroblasts showed elevated aPC production in an in vitro hTM coactivity assay. These findings warrant further work on the control of the xenogenic activation of coagulation by transgenic approaches. [ABSTRACT FROM AUTHOR]

Background: Multi-transgenic pigs produced for use in xenotransplantation have to be screened for the presence and expression of porcine endogenous retroviruses (PERV) to select animals with low PERV load. The production of transgenic pigs may also be associated with the integration of the transgene adjacent to or into the locus of a PERV provirus, potentially leading to an enhanced virus expression. Methods: Non-transgenic animals, single-transgenic, and multi-transgenic pigs were screened for the presence of PERV-A, -B, and -C and recombinant PERV-A/C using polymerase chain reaction (PCR). PERV expression was determined by real time reverse transcriptase-PCR. An assay based on the activation of PERV in peripheral blood mononuclear cells by mitogens was used to discriminate between low and high PERV producer animals. Results: All animals carried PERV-A and -B. A total of 176 from 181 (97.2%) animals carried PERV-C in the germ line and 18 from 64 animals carried PERV-A/C in the genome of lymphoid cells but not in the germ line. The expression of PERV was very low in all animals and not different between transgenic pigs and non-transgenic animals. PERV expression differed between various pig lines. The highest expression was found in mini-pigs and crossing other pig lines with mini-pigs resulted in increased PERV expression in the progeny. However, expression of viral proteins and particle release were not observed in all transgenic animals. Conclusions: No evidence for elevated PERV expression in (multi-) transgenic pigs was observed. Differences in PERV expression correlated with the genetic background of the animals, not with the specific transgene. Mini-pigs consistently had the highest level of PERV expression and animals with a mini-pig background had a higher level of expression compared with animals without mini-pig background. [ABSTRACT FROM AUTHOR]

Background: Xenotransplantation using porcine cells, tissues or organs may be associated with the transmission of porcine endogenous retroviruses (PERVs). More than 50 viral copies have been identified in the pig genome and three different subtypes of PERV were released from pig cells, two of them were able to infect human cells in vitro. RNA interference is a promising option to inhibit PERV transmission. Methods: We recently selected an efficient si (small interfering) RNA corresponding to a highly conserved region in the PERV DNA, which is able to inhibit expression of all PERV subtypes in PERV-infected human cells as well as in primary pig cells. Pig fibroblasts were transfected using a lentiviral vector expressing a corresponding sh (short hairpin) RNA and transgenic pigs were produced by somatic nuclear transfer cloning. Integration of the vector was proven by PCR, expression of shRNA and PERV was studied by in-solution hybridization analysis and real-time RT PCR, respectively. Results: All seven born piglets had integrated the transgene. Expression of the shRNA was found in all tissues investigated and PERV expression was significantly inhibited when compared with wild-type control animals. Conclusion: This strategy may lead to animals compatible with PERV safe xenotransplantation. [ABSTRACT FROM AUTHOR]

Background: Microinjection of foreign DNA into pronuclei of zygotes has been the method of choice for the production of transgenic domestic animals. Following microinjection the transgene is randomly integrated into the host genome which can be associated with insertional mutagenesis and unwanted pathological side effects. Methods: Here, we evaluated the health status of pigs transgenic for the human regulator of complement activation (RCA) CD59 and conducted a complete pathomorphological examination on 19 RCA transgenic pigs at 1 to 32 months of age from nine transgenic lines. Nine wild-type animals served as controls. Expression levels of human complement regulator CD59 (hCD59) mRNA were measured by RT-PCR and distribution of hCD59 protein was determined by immunohistochemistry. Results: Albeit variable transgene expression levels, no specific pathomorphologic phenotype associated with the presence of the transgene in all analyzed pig lines could be detected. Conclusions: Transgenic expression of this human RCA gene construct is not correlated with a specific pathological phenotype in pigs. This is crucial for the application of the technology and the use of transgenic pigs for biomedical and agricultural applications. [ABSTRACT FROM AUTHOR]

Inhibition of complement activation via human membrane-associated complement regulators is known to prevent hyperacute rejection in heart and kidney pig-to-primate transplantation. The protective effect of such strategies in pulmonary xenografts, however, seems to be insufficient. In an ex vivo perfusion, model lungs from donor pigs transgenic for human CD55 ( n = 6) or human CD59 ( n = 5) were perfused with fresh human blood and compared with nontransgenic organs ( n = 6). In addition, a soluble complement component 1 esterase inhibitor (C1-Inh) was applied in h-CD55 transgenic lungs ( n = 3). In the h-CD55 transgenic group, survival was prolonged ( P < 0.05), quality and maximal time of oxygenation significantly improved and pulmonary vascular resistance reduced compared with the control group. There was a decreased sequestration of platelets, less parenchymal injury and reduced deposition of C5b−9 in the h-CD55 transgenic group. Additional soluble complement inhibition (C1-Inh) did not prolong survival of h-CD55 transgenic lungs. Survival and pulmonary function in lungs expressing h-CD59 was not significantly different from parameters observed in nontransgenic lungs. In this ex vivo model of pig-to-primate lung transplantation, membrane-based complement inhibition resulted in significantly improved pulmonary function. However, minor histopathological injuries observed in these transgenic xenografts suggested only partial protection from pulmonary dysfunction by complement inhibition alone. [ABSTRACT FROM AUTHOR]

Microinjection of foreign DNA into pronuclei of a fertilized oocyte has predominantly been used for the generation of transgenic livestock. This technology works reliably, but is inefficient and results in random integration and variable expression patterns in the transgenic offspring. Nevertheless, remarkable achievements have been made with this technology. By targeting expression to the mammary gland, numerous heterologous recombinant human proteins have been produced in large amounts which could be purified from milk of transgenic goats, sheep, cattle and rabbit. Products such as human anti-thrombin III, α-anti-trypsin and tissue plasminogen activator are currently in advanced clinical trials and are expected to be on the market within the next few years. Transgenic pigs that express human complement regulating proteins have been tested in their ability to serve as donors in human organ transplantation (i.e. xenotransplantation). In vitro and in vivo data convincingly show that the hyperacute rejection response can be overcome in a clinically acceptable manner by successful employing this strategy. It is anticipated that transgenic pigs will be available as donors for functional xenografts within a few years. Similarly, pigs may serve as donors for a variety of xenogenic cells and tissues. The recent developments in nuclear transfer and its merger with the growing genomic data allow a targeted and regulatable transgenic production. Systems for efficient homologous recombination in somatic cells are being developed and the adaptation of sophisticated molecular tools, already explored in mice, for transgenic livestock production is underway. The availability of these technologies are essential to maintain “genetic security” and to ensure absence of unwanted side effects. [Copyright &y& Elsevier]

Background: Despite major improvements in pig‐to‐primate xenotransplantation, long‐term survival of xenografts is still challenging. The major histocompatibility complex (MHC) class I, which is crucial in cellular immune response, is an important xenoantigen. Abrogating MHC class I expression on xenografts might be beneficial for extending graft survival beyond current limits. Methods: In this study, we employed the CRISPR/Cas9 system to target exon 2 of the porcine beta‐2‐microglobulin (B2M) gene to abrogate SLA class I expression on porcine cells. B2M‐KO cells served as donor cells for somatic cell nuclear transfer, and cloned embryos were transferred to three recipient sows. The offspring were genotyped for mutations at the B2M locus, and blood samples were analyzed via flow cytometry for the absence of SLA class I molecules. Results: Pregnancies were successfully established and led to the birth of seven viable piglets. Genomic sequencing proved that all piglets carried biallelic modifications at the B2M locus leading to a frameshift, a premature stop codon, and ultimately a functional knockout. However, survival times of these animals did not exceed 4 weeks due to unexpected disease processes. Conclusion: Here, we demonstrate the feasibility of generating SLA class I knockout pigs by targeting the porcine beta‐2‐microglobulin gene using the CRISPR/Cas9 system. Additionally, our findings indicate for the first time that this genetic modification might have a negative impact on the viability of the animals. These issues need to be solved to unveil the real value for xenotransplantation in the future. [ABSTRACT FROM AUTHOR]

Background The acute vascular rejection (AVR) is the major obstacle to successful pig-to-primate xenotransplantation. Changes of the endothelium to a procoagulant state which occur after xenotransplantation entail an increased expression of Tissue Factor (TF). This initiates the extrinsic coagulation cascade and results in microvascular thrombosis and rapid loss of organ function. Organs from pigs with a decreased expression of TF could be promising to overcome AVR. Since a homozygous TF knockout had shown to be lethal in mice [1], we decided to use siRNA-technology to knockdown TF to a basal level. Methods Different siRNAs directed against TF expression were tested in initial studies for their efficacy. A vector coding for the siRNA providing the highest decrease of TF mRNA levels was kindly provided by Dr. Joachim Denner, RKI Berlin. Porcine fetal fibroblasts were co-transfected with this plasmid and a vector (DsRed, Clontech) which confers neomycin resistance and red fluorescence. After 14 days of selection with 800 μg/ml Geneticin (G418), cells were screened for siRNA expression prior to somatic cell nuclear transfers (SCNTs). Subsequently, the reconstructed embryos were transferred to synchronised recipient sows. Liveborn offspring were analysed by real-time PCR for TF mRNA quantification. The production of the siRNA was detected via RT-PCR followed by real-time PCR. Results An average of 95 embryos per sow was transferred to 28 synchronized recipients. The pregnancy rate was 54%. One sow was sacrificed on day 25 of pregnancy and one fetus (#893/1) was obtained. The candidate siRNA could be detected fetal tissues. Real-time PCR analysis revealed decreased TF mRNA expression in fibroblasts of fetus #893/1 compared to wild-type fibroblasts. Therefore, somatic cells from fetus #893/1 was used for recloning. A total of 12 live born piglets could be obtained; five piglets died within few days after delivery. The mRNA levels and production of the siRNA are currently determined. Protein expression analysis by fluorescence cytometry and functionality assessment by coagulation assays are planned for the near future with collaborating laboratories at the Medical School Hannover. Conclusions The AVR is currently the bottleneck in porcine-to-primate organ xenotransplantation. Controlling TF-induced coagulation might provide protective effects for porcine xenografts. Thus, TF knockdown pigs could be critical in producing the ultimate multi-transgenic pig as organ donor for human patients. References [1] Toomey J, Kratzer K, Lasky N, Stanton J, Broze GJ. Targeted disruption of the murine tissue factor gene results in embryonic lethality. Blood. 1996; 88(5): 1583-1587. [ABSTRACT FROM AUTHOR]

Multi-transgenic pigs are required for prolonging survival of porcine xenografts after transplantation into primate recipients. A homozygous knockout of the alpha1,3-galactosyltransferase gene (GGTA-1, encoding for Gal-epitopes) is critical for controlling the hyperacute rejection response. Nevertheless, the additional expression of complement regulatory proteins such as CD55 (decay accelerating factor, DAF) on a GGTA-1 KO background is thought to further improve survival after xenotransplantation. The next hurdle is the acute vascular rejection (AVR), which is primarily caused by endothelial cell activation. Previous experiments had shown that expression of anti-apoptotic/anti-inflammatory genes, such as human heme oxygenase-1 (hHO-1) or human A20 in pig organs may overcome AVR (1, 2). Adult fibroblasts from previously generated hHO-1 transgenic pigs were transfected with Zinc-finger nuclease plasmids directed towards knockout of the GGTA-1 gene. Transfected cells were selected via a magnetic bead selection approach as previously described (3). GGTA-1-KO/hHO-1 cells were used as donor cells in somatic cell nuclear transfer (SCNT). The first pregnancy was terminated at day 25 of gestation and 11 healthy fetuses were obtained. All fetuses were GGTA-1-KO and expressed hHO-1 at similar levels as the hHO-1 transgenic cell donors. Next, fetal GGTA-1-KO/hHO-1 cells from one fetus (C6F5) were transfected with a Sleeping Beauty (SB) transposon (kindly provided by Dr. Zoltan Ivics) coding for hCD55. Two positive cell clones were pooled and used as donor cells for SCNT. One pregnancy was terminated on day 25 and four healthy fetuses were obtained which showed the desired GGTA-1 KO/HO-1/CD55 genotype. Real-time PCR detected a 0.9 fold expression of CD55 over the housekeeping gene GAPDH. Compared to human umbilical vein cells (HUVEC), GGTA-1 KO/HO-1/CD55 fetal fibroblasts expressed CD55 10-15 fold higher than HUVEC. Two fetuses showed a slightly higher expression of CD55 and are currently used as cell donors for recloning to produce live triple transgenic offspring. To solve the problem of transgene segregation occurring after mating of multitransgenic pigs, we generated two tri-cistronic SB vectors coding for A20 and CD55 separated by the self-cleaving peptide E2 site. To select for transfected cells, one vector carried a Tomato-fluorescence cassette whereas the second conferred puromycin resistance. Red fluorescent/puromycin resistant cells served as donor cells for SCNT. Six healthy fetuses were collected on day 25 of pregnancy; all showed high expression of A20 and CD55 as detected by Real-time PCR and FACS. One of the fetuses was used as cell donor for recloning. Currently, three pregnancies with A20/CD55 fetuses are going on and will be allowed to go to term. These results show that molecular scissors, including ZFNs and TALENS, and expression vectors such as the Sleeping beauty system are valuable tools to enhance and accelerate the generation of multi-transgenic pigs for xenotransplantation. References [1] Oropeza M, Petersen B, Carnwath JW et al. Transgenic expression of the human A20 gene in cloned pigs provides protection against apoptotic and inflammatory stimuli. Xenotransplantation. Proc. Nat. Acad. Sci. USA 2009; 16: 522-534. [2] Petersen B, Ramackers W, Lucas-Hahn A et al. Transgenic expression of human heme oxygenase-1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys. Xenotransplantation 2011; in press. [3] Hauschild J, Petersen B, Santiago Y et al. Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases. Proc. Nat. Acad. Sci. USA. 2011; 108: 12013-12017. [ABSTRACT FROM AUTHOR]

Abstract: The shortage of donated human organs for transplantation continues to be a life threatening problem for patients suffering from complete organ failure. Although this gap is increasing due to the demographic changes in aging Western populations, it is generally accepted that international trading in human organ is not an ethical solution. Alternatives to the use of human organs for transplantation must be developed and these alternatives include stem cell therapy, artificial organs and organs from other species, i.e. xenografts. For practical reasons but most importantly because of its physiological similarity with humans, the pig is generally accepted as the species of choice for xenotransplantation. Nevertheless, before porcine organs can be used in human xenotransplantation, it is necessary to make a series of precise genetic modifications to the porcine genome, including the addition of genes for factors which suppress the rejection of transplanted porcine tissues and the inactivation or removal of undesirable genes which can only be accomplished at this time by targeted recombination and somatic nuclear transfer. This review will give an insight into the advances in transgenic manipulation and cloning in pigs—in the context of porcine-to-human xenotransplantation. [Copyright &y& Elsevier]

Abstract: Porcine endogenous retroviruses (PERV) represent a risk for xenotransplantation using pig cells, tissues or organs. PERV-A and PERV-B are present in the genome of all pigs and both infect human cells in vitro. PERV-C infects only pig cells and it is integrated in the genome of most, but not all pigs. Recombinants between PERV-A and PERV-C were described that infect human cells and replicate at high titres. To avoid such recombinations, PERV-C positive animals should not be used for breeding animals suited for xenotransplantation. In order to detect PERV-C positive pigs, different methods were developed such as specific PCRs using different primers, a highly sensitive nested PCR and a real-time PCR allowing measurement of proviral copy numbers. The real-time PCR was found to be useful to discriminate between contamination and actual provirus copies. The PCRs were optimized and their sensitivity was determined. Screening can be started with PCR1, if the result is negative, PCR2 to PCR5 or the nested PCR should be used, if the result is positive, the real-time PCR should be used to exclude contaminations. All methods were used to evaluate the prevalence of PERV-C and to identify PERV-C free animals. Due to the risk of contamination with cells from other animals testing should be performed with blood cells, not with ear biopsies. [Copyright &y& Elsevier]