Article
To read the full-text of this research, you can request a copy directly from the authors.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... Genome editing can be used to alter the DNA in cells, tissues, or model organisms to understand their biology and how they behave in an environment with the altered genome. Genome editing helps in xeno-transplantation, by attempting the transfer of cells, tissues, and organs from animals to treat loss or dysfunction in patients (Perota et al., 2016). Several authors have found CRISPR-Cas9 as an important tool in identifying and characterizing important bacterial virulent genes and elucidating the immune evading strategies adopted by bacteria. ...
... It is likely that stacked gene edits, i.e. edits in more than one gene, will be required to make xenotransplantation practical in a clinical setting. With this in mind, there may be a limit to the number of gene edits that will be compatible with producing a live animal [81,82]. As successes in pig-to-primate xenotransplantation continue to be reported, these preclinical studies should advance progress towards clinical xenotransplantation [82]. ...
Article
Full-text available
Although genetically engineered livestock were first introduced in the 1980s, only three biomedical applications (ATryn ® , Ruconest ® , Kanuma ® ) and one food animal application (AquAdvantage ® salmon) have been brought to market. The initial promise of this technology was hampered by low efficiency, random site integration, public resistance and regulatory hurdles. Gene editing has made it possible to precisely target changes in livestock genomes, overcoming previous challenges. Livestock and poultry editing applications include disease resistance, animal welfare, production traits, animals as bioreactors, xenotransplantation and the development of models for human diseases. The extent to which any of these applications are utilized beyond research laboratories will depend upon regulatory frameworks, uptake by livestock producers and industry, and public acceptance. The 2017 draft US Food and Drug Administration regulatory guidance for gene edited animals, which proposes to regulate ‘intentional’ genomic alterations introduced by editing as new animal drugs irrespective of product novelty or risk, is at odds with emerging regulations around gene edited plants. Lengthy process-based regulation triggered by human ‘intention’ rather than novel product risk may dissuade animal genetics companies and breeders from employing gene editing to introduce beneficial genetic alterations into livestock populations, effectively the same fate encountered by genetic engineering in animals. Goal conflicts will likely continue between genetic innovations to address problems like animal disease and welfare and concerns around the use of modern molecular genetics in food animals. The early applications that successfully navigate regulatory hurdles will influence the public discussion around gene edited animals and impact the trajectory of future applications.
... Genome editing is also helping to transform prospects in xeno-transplantation, by tackling barriers to the transfer of tissues and organs from animals to treat loss or dysfunction in patients (Perota et al., 2016). In particular, genome editing can remove the various xenoreactive animal tissue epitopes, which would otherwise trigger both hyperacute and delayed rejection, and endogenous retroviruses. ...
Article
Full-text available
An EASAC working group on genome editing recommends that regulators should focus on specific applications of these new techniques rather than attempting to regulate genome editing itself as a new technology.
... The advantages offered by genetic engineering to obviate the human immune response may ultimately address the organ shortage [57]. This technology is expected to move xenotransplantation forward more rapidly [54,58]. Cooper et al. commented that "it is likely, therefore, that more rapid progress will be achieved in the genetic manipulation of pigs for the specific purposes of xenotransplantation" [59]. ...
Article
Full-text available
In this article, we examine the advanced clinical development of bioartificial organs and describe the challenges to implementing such systems into patient care. The case for bioartificial organs is evident: they are meant to reduce patient morbidity and mortality caused by the persistent shortage of organs available for allotransplantation. The widespread introduction and adoption of bioengineered organs, incorporating cells and tissues derived from either human or animal sources, would help address this shortage. Despite the decades of development, the variety of organs studied and bioengineered, and continuous progress in the field, only two bioengineered systems are currently commercially available: Apligraf® and Dermagraft® are both approved by the FDA to treat diabetic foot ulcers, and Apligraf® is approved to treat venous leg ulcers. Currently, no products based on xenotransplantation have been approved by the FDA. Risk factors include immunological barriers and the potential infectivity of porcine endogenous retrovirus (PERV), which is unique to xenotransplantation. Recent breakthroughs in gene editing may, however, mitigate risks related to PERV. Because of its primary role in interrupting progress in xenotransplantation, we present a risk assessment for PERV infection, and conclude that the formerly high risk has been reduced to a moderate level. Advances in gene editing, and more broadly in the field, may make it more likely than ever before that bioartificial organs will alleviate the suffering of patients with organ failure.
Article
Full-text available
Gene editing and gene silencing techniques have the potential to revolutionize our knowledge of biology and diseases of fish and other aquatic animals. By using such techniques, it is feasible to change the phenotype and modify cells, tissues and organs of animals in order to cure abnormalities and dysfunctions in the organisms. Gene editing is currently experimental in wide fields of aquaculture, including growth, controlled reproduction, sterility and disease resistance. Zink finger nucleases, TALENs and CRISPR/Cas9 targeted cleavage of the DNA induce favorable changes to site-specific locations. Moreover, gene silencing can be used to inhibit the translation of RNA, namely, to regulate gene expression. This methodology is widely used by researchers to investigate genes involved in different disorders. It is a promising tool in biotechnology and in medicine for investigating gene function and diseases. The production of food fish has increased markedly, making fish and seafood globally more popular. Consequently, the incidence of associated problems and disease outbreaks has also increased. A greater investment in new technologies is therefore needed to overcome such problems in this industry. To put it concisely, the modification of genomic DNA and gene silencing can comprehensively influence aquatic animal medicine in the future. On the ethical side, these precise genetic modifications make it more complicated to recognize genetically modified organisms in nature and can cause several side effects through created mutations. The aim of this review is to summarize the current state of applications of gene modifications and genome editing in fish medicine.
Article
Full-text available
The rapid advancement in targeted genome editing using engineered nucleases such as ZFNs, TALENs and CRISPR/Cas9 systems has resulted in a suite of powerful methods that allows researchers to target any genomic locus of interest. A complementary set of design tools has been developed to aid researchers with nuclease design, target site selection, and experimental validation. Here we review the various tools available for target selection in designing engineered nucleases, and for quantifying nuclease activity and specificity, including web-based search tools and experimental methods. We also elucidate challenges in target selection, especially in predicting off-target effects, and discuss future directions in precision genome editing and its applications.Molecular Therapy (2016); doi:10.1038/mt.2016.1.
Article
Full-text available
Targeted modification of the pig genome can be challenging. Recent applications of the CRISPR/Cas9 system hold promise for improving the efficacy of genome editing. When a designed CRISPR/Cas9 system targeting CD163 or CD1D was introduced into somatic cells, it was highly efficient in inducing mutations. When these mutated cells were used with somatic cell nuclear transfer, offspring with these modifications were created. When the CRISPR/Cas9 system was delivered into in vitro produced presumptive porcine zygotes, the system was effective in creating mutations in eGFP, CD163 and CD1D (100% targeting efficiency in blastocyst stage embryos); however, it also presented some embryo toxicity. We could also induce deletions in CD163 or CD1D by introducing two types of CRISPRs with Cas9. The system could also disrupt two genes, CD163 and eGFP, simultaneously when two CRISPRs targeting two genes with Cas9 were delivered into zygotes. Direct injection of CRISPR/Cas9 targeting CD163 or CD1D into zygotes resulted in piglets that have mutations on both alleles with only one CD1D pig having a mosaic genotype. We show here that the CRISPR/Cas9 system can be used by two methods. The system can be used to modify somatic cells followed by somatic cell nuclear transfer. System components can also be used in in vitro produced zygotes to generate pigs with specific genetic modifications.
Article
Full-text available
Cell death and differentiation is a monthly research journal focused on the exciting field of programmed cell death and apoptosis. It provides a single accessible source of information for both scientists and clinicians, keeping them up-to-date with advances in the field. It encompasses programmed cell death, cell death induced by toxic agents, differentiation and the interrelation of these with cell proliferation.
Article
Full-text available
Inbred mini-pigs are ideal organ donors for future human xenotransplantations because of their clear genetic background, high homozygosity, and high inbreeding endurance. In this study, we chose fibroblast cells from a highly inbred pig line called Banna mini-pig inbred line (BMI) as donor nuclei for nuclear transfer, combining with transcription activator-like effector nucleases (TALENs) and successfully generated α-1,3-galactosyltransferase (GGTA1) gene biallelic knockout (KO) pigs. To validate the efficiency of TALEN vectors, in vitro-transcribed TALEN mRNAs were microinjected into one-cell stage parthenogenetically activated porcine embryos. The efficiency of indel mutations at the GGTA1-targeting loci was as high as 73.1% (19/26) among the parthenogenetic blastocysts. TALENs were co-transfected into porcine fetal fibroblasts of BMI with a plasmid containing neomycin gene. The targeting efficiency reached 89.5% (187/209) among the survived cell clones after a 10 d selection. More remarkably 27.8% (58/209) of colonies were biallelic KO. Five fibroblast cell lines with biallelic KO were chosen as nuclear donors for somatic cell nuclear transfer (SCNT). Three miniature piglets with biallelic mutations of the GGTA1 gene were achieved. Gal epitopes on the surface of cells from all the three biallelic KO piglets were completely absent. The fibroblasts from the GGTA1 null piglets were more resistant to lysis by pooled complement-preserved normal human serum than those from wild-type pigs. These results indicate that a combination of TALENs technology with SCNT can generate biallelic KO pigs directly with high efficiency. The GGTA1 null piglets with inbred features created in this study can provide a new organ source for xenotransplantation research.
Article
Full-text available
Acellular materials of xenogenic origin are used worldwide as xenografts, and phase I trials of viable pig pancreatic islets are currently being performed. However, limited information is available on transmission of porcine endogenous retrovirus (PERV) after xenotransplantation and on the long-term immune response of recipients to xenoantigens. We analyzed the blood of burn patients who had received living pig-skin dressings for up to 8 wk for the presence of PERV as well as for the level and nature of their long term (maximum, 34 y) immune response against pig Ags. Although no evidence of PERV genomic material or anti-PERV Ab response was found, we observed a moderate increase in anti-αGal Abs and a high and sustained anti-non-αGal IgG response in those patients. Abs against the nonhuman sialic acid Neu5Gc constituted the anti-non-αGal response with the recognition pattern on a sialoglycan array differing from that of burn patients treated without pig skin. These data suggest that anti-Neu5Gc Abs represent a barrier for long-term acceptance of porcine xenografts. Because anti-Neu5Gc Abs can promote chronic inflammation, the long-term safety of living and acellular pig tissue implants in recipients warrants further evaluation.
Article
Full-text available
After the knock-out (KO) of α1,3 galactosyltransfease (Gal-T), the Hanganutziu-Deicher antigen became a major antigen of the "non-Gal antigen" that is implicated in subsequent xenograft rejection. For deletion of non-Gal antigen, we successfully produced zinc finger nuclease (ZFN)-mediated monoallelic/biallelic male and female CMP-N-acetylneuraminic acid hydroxylase (CMAH) KO miniature pigs: the efficiency of the gene targeting (41.7%) was higher when donor DNA was used with the ZFN than those of ZFN alone (9.1%). Monoallelic KO pigs had no integration of exogenous DNA into their genome, indicating that this technique would provide a new avenue to reduce the risk of antibiotics resistance when organs from genetically modified pigs are transplanted into patients. Until now, both monoallelic and biallelic CMAH KO pigs are healthy and show no sign of abnormality and off-target mutations. Therefore, these CMAH null pigs on the Gal-T KO background could serve as an important model for the xenotransplantation.
Article
Full-text available
In the field of regenerative medicine, one of the ultimate goals is to generate functioning organs from pluripotent cells, such as ES cells or induced pluripotent stem cells (PSCs). We have recently generated functional pancreas and kidney from PSCs in pancreatogenesis- or nephrogenesis-disabled mice, providing proof of principle for organogenesis from PSCs in an embryo unable to form a specific organ. Key when applying the principles of in vivo generation to human organs is compensation for an empty developmental niche in large nonrodent mammals. Here, we show that the blastocyst complementation system can be applied in the pig using somatic cell cloning technology. Transgenic approaches permitted generation of porcine somatic cell cloned embryos with an apancreatic phenotype. Complementation of these embryos with allogenic blastomeres then created functioning pancreata in the vacant niches. These results clearly indicate that a missing organ can be generated from exogenous cells when functionally normal pluripotent cells chimerize a cloned dysorganogenetic embryo. The feasibility of blastocyst complementation using cloned porcine embryos allows experimentation toward the in vivo generation of functional organs from xenogenic PSCs in large animals.
Article
Full-text available
The possible use of pig organs and tissues as xenografts in humans is actively being considered in biomedical research. We therefore examined whether pig endogenous retrovirus (PERV) genomes can be infectiously transmitted to human cells in culture. Two pig kidney cell lines spontaneously produce C-type retrovirus particles. Cell-free retrovirus produced by the PK-15 kidney cell line (PERV-PK) infected pig, mink and human kidney 293 cell lines and co-cultivation of X-irradiated PK-15 cells with human cells resulted in a broader range of human cell infection, including human diploid fibroblasts and B- and T-cell lines. Kidney, heart and spleen tissue obtained from domestic pigs contained multiple copies of integrated PERV genomes and expressed viral RNA. Upon passage in human cells PERV-PK could rescue a Moloney retroviral vector and acquired resistance to lysis by human complement.
Article
ntroduction The process of xenograft rejection in a pig to baboon large animal cardiac xenotransplantation model is very complex and therefore an equally multifaceted treatment is required to overcome this hurdle. Here we describe a successful strategy to subdue the strong xenogeneic immune response and achieve prolong graft survival. Material and Methods Five 6–8 week old piglets which were genetically engineered to remove alpha 1–3 galactosyltransferase gene and expressed human CD46 and thrombomodulin transgenes, served as heterotopic heart donors. Size matched specific pathogen free baboons were used as the recipients (n = 5). Immunomodulation consisted of B cell depletion with four doses of anti CD20 (19 mg/kg) (days −7, 0, 7, 14) in the induction phase, immune suppression with mycophenolate mofitel (20 mg/kg) daily and steroids (2 mg/kg) (tapered off in 2 months), and co-stimulation blockade with high dose anti CD40 antibody (Clone 2C10R4) (50 mg/kg) on days −1, 0, 5, 9, 14 then weekly. The dose of anti CD40 antibody was first reduced in two baboons on day 100, in two baboons on day 365 (in these baboons, anti CD40 antibody treatment was tapered off and was completely stopped on day 560 in one and day 861 in another) and in one baboon the dose was not reduced. Results Significant graft survival was achieved in all 5 baboons. In two baboons in which antibody was reduced (25 mg/Kg) on day 100, grafts were rejected within 290 days while the baboons in which antibody was tapered after 1 year, grafts survived for the longest period of time (616 and 945 days). One baboon where dose reduction did not occur developed an antibiotic resistant infection and rejected the graft on day 146. The most interesting finding was that the two long-term (>600 days) surviving hearts rejected within 12 weeks of discontinuing anti CD40 antibody therapy indicating the dependence of graft survival on the anti CD40 antibody treatment. The period taken for these hearts to reject after cessation of anti CD40 antibody treatment also coincided with the time for complete washout of this antibody from the recipient's blood circulation. Conclusion Our results clearly indicate that, for long-term xenograft survival, genetically engineered donor pig hearts and high dose of anti CD40 antibody are required to avoid; antibody mediated rejection, complement activation and thrombotic coagulopathies. However, it is also clear that dose and duration of anti CD40 antibody treatment had a critical impact on maintenance of graft survival.
Article
Although the expression of endogenous retroviruses in the placenta of numerous species was observed a long time ago, their physiological function during gestation was demonstrated only very recently. Expression of retroviral envelope proteins, also called syncytins, in the placenta allows generation of the multinuclear syncytiotrophoblast as an outer cellular layer of the placenta by fusion of the trophoblast cells. This fusion process is crucial for the development of the placenta and for successful pregnancy. It is still unclear whether the immunosuppressive properties of the transmembrane envelope protein of the endogenous retroviruses expressed in the placenta contribute to immunosuppression to prevent the rejection of the semiallotransplant embryo. The presence of placenta cells expressing retroviral envelope proteins surrounded by immune cells deep in the maternal tissue supports an immunosuppressive function. It is important to emphasize that during evolution different species utilized (‘enslaved’) different endogenous retroviruses and that two or more endogenous retroviruses are involved in placentogenesis in each species.
Article
Virally cleansing the pig genome Transplants from pigs could be a solution to a shortage of human organs for transplantation. Unfortunately, porcine endogenous retroviruses (PERVs) are rife in pigs and can be transmitted to humans, risking disease. L. Yang et al. integrated CRISPR-Cas into the pig cell genome, where continuous induction of the Cas9 editing enzyme resulted in the mutation of every single PERV reverse transcriptase gene. This prevented replication of all copies of PERV, viral infection, and transmission to human cells. Science , this issue p. 1101
Article
Acute vascular rejection (AVR), in particular microvascular thrombosis, is an important barrier to successful pig-to-primate xenotransplantation. Here, we report the generation of pigs with decreased tissue factor (TF) levels induced by small interfering (si)RNA-mediated gene silencing. Porcine fibroblasts were transfected with TF-targeting small hairpin (sh)RNA and used for somatic cell nuclear transfer. Offspring were analyzed for siRNA, TF mRNA and TF protein level. Functionality of TF downregulation was investigated by a whole blood clotting test and a flow chamber assay. TF siRNA was expressed in all twelve liveborn piglets. TF mRNA expression was reduced by 94.1 ± 4.7% in TF knockdown (TFkd) fibroblasts compared to wild-type (WT). TF protein expression in PAEC stimulated with 50 ng/mL TNF-α was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 ± 136 vs. WT: 13 038 ± 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 ± 8.8 min, WT: 45.8 ± 7.7 min, p < 0.0001) and significantly decreased thrombus formation compared to WT (mean thrombus coverage per viewing field in %; WT: 23.5 ± 13.0, TFkd: 2.6 ± 3.7, p < 0.0001). Our data show that a functional knockdown of TF is compatible with normal development and survival of pigs. TF knockdown could be a valuable component in the generation of multi-transgenic pigs for xenotransplantation. © Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.
Article
Background Simultaneous inactivation of pig GGTA1 and CMAH genes eliminates carbohydrate xenoantigens recognized by human antibodies. The β4GalNT2 glycosyltransferase may also synthesize xenoantigens. To further characterize glycan-based species incompatibilities, we examined human and non-human primate antibody binding to cells derived from genetically modified pigs lacking these carbohydrate-modifying genes.Methods The Cas9 endonuclease and gRNA were used to create pigs lacking GGTA1, GGTA1/CMAH, or GGTA1/CMAH/β4GalNT2 genes. Peripheral blood mononuclear cells were isolated from these animals and examined for binding to IgM and IgG from humans, rhesus macaques, and baboons.ResultsCells from GGTA1/CMAH/β4GalNT2 deficient pigs exhibited reduced human IgM and IgG binding compared to cells lacking both GGTA1 and CMAH. Non-human primate antibody reactivity with cells from the various pigs exhibited a slightly different pattern of reactivity than that seen in humans. Simultaneous inactivation of the GGTA1 and CMAH genes increased non-human primate antibody binding compared to cells lacking either GGTA1 only or to those deficient in GGTA1/CMAH/β4GalNT2.Conclusions Inactivation of the β4GalNT2 gene reduces human and non-human primate antibody binding resulting in diminished porcine xenoantigenicity. The increased humoral immunity of non-human primates toward GGTA1-/CMAH-deficient cells compared to pigs lacking either GGTA1 or GGTA1/CMAH/β4GalNT2 highlights the complexities of carbohydrate xenoantigens and suggests potential limitations of the non-human primate model for examining some genetic modifications. The progressive reduction of swine xenoantigens recognized by human immunoglobulin through inactivation of pig GGTA1/CMAH/β4GalNT2 genes demonstrates that the antibody barrier to xenotransplantation can be minimized by genetic engineering.
Article
Current technology enables the production of highly specific genome modifications with excellent efficiency and specificity. Key to this capability are targetable DNA cleavage reagents and cellular DNA repair pathways. The break made by these reagents can produce localized sequence changes through inaccurate nonhomologous end joining (NHEJ), often leading to gene inactivation. Alternatively, user-provided DNA can be used as a template for repair by homologous recombination (HR), leading to the introduction of desired sequence changes. This review describes three classes of targetable cleavage reagents: zincfinger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas RNA-guided nucleases (RGNs). As a group, these reagents have been successfully used to modify genomic sequences in a wide variety of cells and organisms, including humans. This review discusses the properties, advantages, and limitations of each system, as well as the specific considerations required for their use in different biological systems. Expected final online publication date for the Annual Review of Biochemistry Volume 83 is June 02, 2014. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Article
The discrepancy between organ need and organ availability represents one of the major limitations in the field of transplantation. One possible solution to this problem is xenotransplantation. Research in this field has identified several obstacles that have so far prevented the successful development of clinical xenotransplantation protocols. The main immunologic barriers include strong T-cell and B-cell responses to solid organ and cellular xenografts. In addition, components of the innate immune system can mediate xenograft rejection. Here, we review these immunologic and physiologic barriers and describe some of the strategies that we and others have developed to overcome them. We also describe the development of two strategies to induce tolerance across the xenogeneic barrier, namely thymus transplantation and mixed chimerism, from their inception in rodent models through their current progress in preclinical large animal models. We believe that the addition of further beneficial transgenes to Gal knockout swine, combined with new therapies such as Treg administration, will allow for successful clinical application of xenotransplantation.
Article
Clinical xenotransplantation is not possible because humans possess antibodies that recognize antigens on the surface of pig cells. Galα-1,3-Gal (Gal) and N-glycolylneuraminic acid (Neu5Gc) are two known xenoantigens. We report the homozygous disruption of the α1, 3-galactosyltransferase (GGTA1) and the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes in liver-derived female pig cells using zinc-finger nucleases (ZFNs). Somatic cell nuclear transfer (SCNT) was used to produce healthy cloned piglets from the genetically modified liver cells. Antibody-binding and antibody-mediated complement-dependent cytotoxicity assays were used to examine the immunoreactivity of pig cells deficient in Neu5Gc and Gal. This approach enabled rapid production of a pig strain deficient in multiple genes without extensive breeding protocols. Immune recognition studies showed that pigs lacking both CMAH and GGTA1 gene activities reduce the humoral barrier to xenotransplantation, further than pigs lacking only GGTA1. This technology will accelerate the development of pigs for xenotransplantation research.
Article
Because a shortage of donor organs has been a major obstacle to the expansion of organ transplantation programs, the generation of transplantable organs is among the ultimate goals of regenerative medicine. However, the complex cellular interactions among and within tissues that are required for organogenesis are difficult to recapitulate in vitro. As an alternative, we used blastocyst complementation to generate pluripotent stem cell (PSC)-derived donor organs in vivo. We hypothesized that if we injected PSCs into blastocysts obtained from mutant mice in which the development of a certain organ was precluded by genetic manipulation, thereby leaving a niche for organ development, the PSC-derived cells would developmentally compensate for the defect and form the missing organ. In our previous work, we showed proof-of-principle findings of pancreas generation by injection of PSCs into pancreas-deficient Pdx1(-/-) mouse blastocysts. In this study, we have extended this technique to kidney generation using Sall1(-/-) mouse blastocysts. As a result, the defective cells were totally replaced, and the kidneys were entirely formed by the injected mouse PSC-derived cells, except for structures not under the influence of Sall1 expression (ie, collecting ducts and microvasculature). These findings indicate that blastocyst complementation can be extended to generate PSC-derived kidneys. This system may therefore provide novel insights into renal organogenesis.
Article
Fishman JA, Scobie L, Takeuchi Y. Xenotransplantation-associated infectious risk: a WHO consultation. Xenotransplantation 2012; 19: 72–81. © 2012 John Wiley & Sons A/S. Abstract: Xenotransplantation carries the potential risk of the transmission of infection with the cells or tissues of the graft. The degree of risk is unknown in the absence of clinical trials. The clinical application of xenotransplantation has important implications for infectious disease surveillance, both at the national and international levels. Preclinical data indicate that infectious disease events associated with clinical xenotransplantation from swine, should they occur, will be rare; data in human trials are limited but have demonstrated no transmission of porcine microorganisms including porcine endogenous retrovirus. Xenotransplantation will necessitate the development of surveillance programs to detect known infectious agents and, potentially, previously unknown or unexpected pathogens. The development of surveillance and safety programs for clinical trials in xenotransplantation is guided by a “Precautionary Principle,” with the deployment of appropriate screening procedures and assays for source animals and xenograft recipients even in the absence of data suggesting infectious risk. All assays require training, standardization and validation, and sharing of laboratory methods and expertise to optimize the quality of the surveillance and diagnostic testing. Investigation of suspected xenogeneic infection events (xenosis, xenozoonosis) should be performed in collaboration with an expert data safety review panel and the appropriate public health and competent authorities. It should be considered an obligation of performance of xenotransplantation trials to report outcomes, including any infectious disease transmissions, in the scientific literature. Repositories of samples from source animals and from recipients prior to, and following xenograft transplantation are essential to the investigation of possible infectious disease events. Concerns over any potential hazards associated with xenotransplantation may overshadow potential benefits. Careful microbiological screening of source animals used as xenotransplant donors may enhance the safety of transplantation beyond that of allotransplant procedures. Xenogeneic tissues may be relatively resistant to infection by some human pathogens. Moreover, xenotransplantation may be made available at the time when patients require organ replacement on a clinical basis. Insights gained in studies of the microbiology and immunology of xenotransplantation will benefit transplant recipients in the future. This document summarizes approaches to disease surveillance in individual recipients of nonhuman tissues.
Article
To investigate the hypothesis that the human endogenous sequence ERV-3 has a function, we have cloned and expressed the transmembrane region of its envelope gene and raised specific antibodies to the fusion protein and to a synthetic peptide. These antibodies reacted with a 65-kDa polypeptide which constituted approximately 0.1% of the cellular protein in syncytiotrophoblasts in placenta. The evolutionary conservation and abundant expression of this endogenous retroviral protein in a specific cell type support the concept of a biological function. The similarity of a domain of ERV-3 env to putative immunosuppressive p15E sequences suggests that ERV-3 might form part of the placental immunosuppressive barrier between mother and foetus.
Article
Galactosyl-transferase knockout (GT-KO) pigs represent the latest major progress to reduce immune reactions in xenotransplantation. However, their organs are still subject to rapid humoral rejection involving complement activation requiring the ongoing development of further genetic modifications in the pig. In a pig-to-baboon renal transplantation setting, we have used donor pigs that are not only GT-KO, but also transgenic for human CD55 (hCD55), hCD59, hCD39, and fucosyl-transferase (hHT). We studied kidney xenograft survival, physiological and immunologic parameters, xenogeneic rejection characteristics, as well as viral transmission aspects among two groups of baboons: control animals (n = 2), versus those (n = 4) treated with a cocktail of cyclophosphamide, tacrolimus, mycophenolate mofetil, steroids, and a recombinant human C1 inhibitor. Whereas control animals showed clear acute humoral rejection at around day 4, the treated animals showed moderately improved graft survival with rejection at around 2 weeks posttransplantation. Biopsies showed signs of acute vascular rejection (interstitial hemorrhage, glomerular thrombi, and acute tubular necrosis) as well as immunoglobulin (Ig)M and complement deposition in the glomerular and peritubular capillaries. The low level of preformed non-Gal-α1.3Gal IgM detected prior to transplantation increased at 6 days posttransplantation, whereas induced IgG appeared after day 6. No porcine endogenous retrovirus (PERV) transmission was detected in any transplanted baboon. Thus, surprisingly, organs from the GT-KO, hCD55, hCD59, hCD39, and hHT transgenic donors did not appear to convey significant protection against baboon anti-pig antibodies and complement activation, which obviously continue to be significant factors under a suboptimal immunosuppression regimen. The association, timing, and doses of immunosuppressive drugs remain critical. They will have to be optimized to achieve longer graft survivals.
Article
The shortage of organs and cells from deceased individuals continues to restrict allotransplantation. Pigs could provide an alternative source of tissue and cells but the immunological challenges and other barriers associated with xenotransplantation need to be overcome. Transplantation of organs from genetically modified pigs into non-human primates is now not substantially limited by hyperacute, acute antibody-mediated, or cellular rejection, but other issues have become more prominent, such as development of thrombotic microangiopathy in the graft or systemic consumptive coagulopathy in the recipient. To address these problems, pigs that express one or more human thromboregulatory or anti-inflammatory genes are being developed. The results of preclinical transplantation of pig cells--eg, islets, neuronal cells, hepatocytes, or corneas--are much more encouraging than they are for organ transplantation, with survival times greater than 1 year in all cases. Risk of transfer of an infectious microorganism to the recipient is small.
Article
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.
Article
The complexity of organogenesis hinders in vitro generation of organs derived from a patient's pluripotent stem cells (PSCs), an ultimate goal of regenerative medicine. Mouse wild-type PSCs injected into Pdx1(-/-) (pancreatogenesis-disabled) mouse blastocysts developmentally compensated vacancy of the pancreatic "developmental niche," generating almost entirely PSC-derived pancreas. To examine the potential for xenogenic approaches in blastocyst complementation, we injected mouse or rat PSCs into rat or mouse blastocysts, respectively, generating interspecific chimeras and thus confirming that PSCs can contribute to xenogenic development between mouse and rat. The development of these mouse/rat chimeras was primarily influenced by host blastocyst and/or foster mother, evident by body size and species-specific organogenesis. We further injected rat wild-type PSCs into Pdx1(-/-) mouse blastocysts, generating normally functioning rat pancreas in Pdx1(-/-) mice. These data constitute proof of principle for interspecific blastocyst complementation and for generation in vivo of organs derived from donor PSCs using a xenogenic environment.
Xenotransplantation of galactosyl-transferase knockout, CD55, CD59, CD39, and fucosyltransferase transgenic pig kidneys into baboons
  • Le Bas-Bernardet
  • S Tillou
  • X Poirier
  • N Dilek
  • N Chatelais
  • M Devalliere
  • J Charreau
  • B Minault
  • D Hervouet
  • J Renaudin
  • K Crossan
  • C Scobie
  • L Cowan
  • P J Apice
  • A J Galli
  • C Cozzi
  • E Soulillou
  • J P Vanhove
  • B Blancho
Le Bas-Bernardet, S., Tillou, X., Poirier, N., Dilek, N., Chatelais, M., Devalliere, J., Charreau, B., Minault, D., Hervouet, J., Renaudin, K., Crossan, C., Scobie, L., Cowan, P.J., d'Apice, A.J., Galli, C., Cozzi, E., Soulillou, J.P., Vanhove, B., Blancho, G., 2011. Xenotransplantation of galactosyl-transferase knockout, CD55, CD59, CD39, and fucosyltransferase transgenic pig kidneys into baboons. Transplant. Proc. 43, 3426e3430.
Long-term IgG response to porcine Neu5Gc antigens without transmission of PERV in burn patients treated with porcine skin xenografts
  • L Scobie
  • V Padler-Karavani
  • Le Bas-Bernardet
  • S Crossan
  • C Blaha
  • J Matouskova
  • M Hector
  • R D Cozzi
  • E Vanhove
  • B Charreau
  • B Blancho
  • G Bourdais
  • L Tallacchini
  • M Ribes
  • J M Yu
  • H Chen
  • X Kracikova
  • J Broz
  • L Hejnar
  • J Vesely
  • P Takeuchi
  • Y Varki
  • A Soulillou
Scobie, L., Padler-Karavani, V., Le Bas-Bernardet, S., Crossan, C., Blaha, J., Matouskova, M., Hector, R.D., Cozzi, E., Vanhove, B., Charreau, B., Blancho, G., Bourdais, L., Tallacchini, M., Ribes, J.M., Yu, H., Chen, X., Kracikova, J., Broz, L., Hejnar, J., Vesely, P., Takeuchi, Y., Varki, A., Soulillou, J.P., 2013. Long-term IgG response to porcine Neu5Gc antigens without transmission of PERV in burn patients treated with porcine skin xenografts. J. Immunol. 191, 2907e2915.
  • L Yang
  • M Guell
  • D Niu
  • H George
  • E Lesha
  • D Grishin
  • J Aach
  • E Shrock
  • W Xu
  • J Poci
  • R Cortazio
  • R A Wilkinson
  • J A Fishman
  • G Church
Yang, L., Guell, M., Niu, D., George, H., Lesha, E., Grishin, D., Aach, J., Shrock, E., Xu, W., Poci, J., Cortazio, R., Wilkinson, R.A., Fishman, J.A., Church, G., 2015. Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science 350, 1101e1104.