Porcine Endogenous Retrovirus Transmission Research Articles

Porcine endogenous retroviruses in xenotransplantation.

Xenotransplantation using pig cells, tissues or organs is under development to alleviate the shortage of human donor organs. Meanwhile, remarkably long survival times of pig organs in non-human primates have been reported, as well as the functionality of pig kidneys and hearts in brain-dead humans. Most importantly, two transplantations of pig hearts in patients were performed with survival times of the patients of 8 and 6 weeks. Xenotransplantation may be associated with the transmission of porcine microorganisms including viruses to the recipient. Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pigs and cannot be eliminated like other viruses can. PERVs are able to infect certain human cells and therefore pose a risk for xenotransplantation. It is well known that retroviruses are able to induce tumors and immunodeficiencies. However, until now, PERVs were not transmitted in all infection experiments using small animals and non-human primates, in all preclinical xenotransplantation trials in non-human primates and in all clinical trials in humans. In addition, several strategies including antiretrovirals, PERV-specific small interfering RNA, vaccines and genome editing using CRISPR/Cas have been developed to prevent PERV transmission.

Analysis of PERV-C superinfection resistance using HA-tagged viruses

BackgroundUsing pigs as organ donors has advanced xenotransplantation to the point that it is almost ready for clinical use. However, there is still a zoonotic risk associated with xenotransplantation, and the potential transmission of porcine endogenous retroviruses needs to be surveyed. Despite significant attempts to eliminate this risk, by the selection of PERV-C free pigs with low expression of PERV-A, -B, and by the genome-wide inactivation of PERV using CRISPR/Cas9, the impact of superinfection resistance (SIR) was not investigated. SIR is a viral trait that prevents reinfection (superinfection). For PERV, the underlying mechanism is unclear, whether and how cells, that harbor functional PERV, are protected. Using PERV-C(5683) as a reference virus, we investigated SIR in a newly developed in vitro model to pursue the mechanism and confirm its protective effect.ResultsWe developed three PERV-C constructs on the basis of PERV-C(5683), each of which carries a hemagglutinin tag (HA-tag) at a different position of the envelope gene (SP-HA, HA-VRA, and RPep-HA), to distinguish between primary infection and superinfection. The newly generated PERV-C(5683)-HA viruses were characterized while quantifying the viral RNA, reverse transcriptase activity, protein expression analysis, and infection studies. It was demonstrated that SP-HA and RPep-HA were comparable to PERV-C(5683), whereas HA-VRA was not replication competent. SP-HA and RPep-HA were chosen to challenge PERV-C(5683)-positive ST-IOWA cells demonstrating that PERV-C-HA viruses are not able to superinfect those cells. They do not integrate into the genome and are not expressed.ConclusionsThe mechanism of SIR applies to PERV-C. The production of PERV-C particles serves as a defense mechanism from superinfection with exogenous PERV-C. It was demonstrated by newly generated PERV-C(5683)-HA clones that might be used as a cutting-edge tool. The HA-tagging of PERV-C is novel, providing a blueprint for the tagging of other human tropic PERV viruses. The tagged viruses are suitable for additional in vitro and in vivo infection studies and will contribute, to basic research on viral invasion and pathogenesis. It will maintain the virus safety of XTx.

Porcine Endogenous Retroviruses and Xenotransplantation, 2021

Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pigs, and some of them are able to infect human cells. Therefore, PERVs pose a risk for xenotransplantation, the transplantation of pig cells, tissues, or organ to humans in order to alleviate the shortage of human donor organs. Up to 2021, a huge body of knowledge about PERVs has been accumulated regarding their biology, including replication, recombination, origin, host range, and immunosuppressive properties. Until now, no PERV transmission has been observed in clinical trials transplanting pig islet cells into diabetic humans, in preclinical trials transplanting pig cells and organs into nonhuman primates with remarkable long survival times of the transplant, and in infection experiments with several animal species. Nevertheless, in order to prevent virus transmission to the recipient, numerous strategies have been developed, including selection of PERV-C-free animals, RNA interference, antiviral drugs, vaccination, and genome editing. Furthermore, at present there are no more experimental approaches to evaluate the full risk until we move to the clinic.

Solid organ xenotransplantation at the interface between research and clinical development: Regulatory aspects.

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

Detection of Pig Cells Harboring Porcine Endogenous Retroviruses in Non-Human Primate Bladder After Renal Xenotransplantation.

Pigs are used as potential donor animals for xenotransplantation. However, porcine endogenous retrovirus (PERV), shown to infect both human and non-human primate (NHP) cells in vitro, presents a risk of transmission to humans in xenotransplantation. In this study, we analyzed PERV transmission in various organs after pig-to-NHP xenotransplantation. We utilized pig-to-NHP xenotransplant tissue samples obtained using two types of transgenic pigs from the National Institute of Animal Science (NIAS, Republic of Korea), and examined them for the existence of PERV genes in different organs via PCR and RT-PCR with specific primers. To determine PERV insertion into chromosomes, inverse PCR using PERV long terminal repeat (LTR) region-specific primers was conducted. The PERV gene was not detected in NHP organs in cardiac xenotransplantation but detected in NHP bladders in renal xenotransplantation. The insertion experiment confirmed that PERVs originate from porcine donor cells rather than integrated provirus in the NHP chromosome. We also demonstrate the presence of pig cells in the NHP bladder after renal xenotransplantation using specific-porcine mitochondrial DNA gene PCR. The PERV sequence was detected in the bladder of NHPs after renal xenotransplantation by porcine cell-microchimerism but did not integrate into the NHP chromosome.

Expression profile of human porcine endogenous retrovirus Areceptors (HuPAR-1, HuPAR-2) and transcription factor activator protein-2γ (TFAP-2C) genes in infected human fibroblasts-Model in vitro.

Xenotransplantation of porcine tissues raises concerns, especially in the context of the potential interspecies transmission of porcine endogenous retroviruses (PERVs). To date, the possibility of PERV infections of various human cells has been confirmed in vitro. PERVs infect cells coupling viral Env protein with adequate functional receptor on the surface of the host cell. So far, two PERV-A receptors have been described in humans: HuPAR-1 and HuPAR-2. TFAP-2C was described as one of the transcription factors engaged in the expression of HuPAR-2. Bacterial LPS, well known as a strong inflammation inducer, was used in this study to stimulate changes of the expression profile of inflammation-related genes in human cells in vitro. The aim of the study was to investigate the expression profile of HuPAR-1 and HuPAR-2 and TFAP-2C genes in human NHDF cells treated with LPS and/or infected with PERVs from PK15 cells. PERV infection and expression was confirmed by qPCR and RTqPCR. The expression of HuPAR-1, HuPAR-2, and TFAP-2C genes was studied using HGU 133A 2.0 microarrays and RTqPCR. NHDF cells expressed both HuPAR-1 and HuPAR-2 genes with a higher expression of HuPAR-1. LPS down-regulated the expression of HuPAR-1 and TFAP-2C in NHDF cells, but had no effect on HuPAR-2 expression. These changes induced by LPS were more pronounced in the presence of PERV infection. As reported previously, treatment of NHDF cells with LPS decreased PERV-A provirus integration and increased PERV-A mRNA expression. PERV infection alone did not modulate the expression of HuPAR-1, HuPAR-2, and TFAP-2C. This is the first study analyzing the expression profile of HuPAR-1, HuPAR-2, and TFAP-2C in NHDF cells treated by LPS and/or infected by PERVs.

Reducing porcine corneal graft rejection, with an emphasis on porcine endogenous retrovirus transmission safety: a review.

Donor cornea shortage is a primary hurdle in the development of corneal transplantation. Of all species, porcine corneas are the ideal transplantation material for humans. However, the xenoimmune rejection induced by porcine corneal xenotransplantation compromises surgical efficacy. Although the binding of IgM/IgG in human serum to a genetically modified porcine cornea is significantly weaker than that of the wild type (WT), genetically modified porcine corneas do not display a prolonged graft survival time in vivo. Conversely, costimulatory blockade drugs, such as anti-CD40 antibodies, can reduce the xenoimmune response and prolong graft survival time in animal experiments. Moreover, porcine endothelial grafts can survive for more than 6mo with only the subconjunctival injection of a steroid-based immunosuppressants regime; therefore, they show great value for treating corneal endothelial disease. In addition, zoonotic transmission is a primary concern of xenotransplantation. Porcine endogenous retrovirus (PERV) is the most significant virus assessed by ophthalmologists. PERV integrates into the porcine genome and infects human cells in vitro. Fortunately, no evidence from in vivo studies has yet shown that PERV can be transmitted to hosts.

Human SAMHD1 restricts the xenotransplantation relevant porcine endogenous retrovirus (PERV) in non-dividing cells.

The release of porcine endogenous retrovirus (PERV) particles from pig cells is a potential risk factor during xenotransplantation by way of productively infecting the human transplant recipient. Potential countermeasures against PERV replication are restriction factors that block retroviral replication. SAMHD1 is a triphosphohydrolase that depletes the cellular pool of dNTPs in non-cycling cells starving retroviral reverse transcription. We investigated the antiviral activity of human SAMHD1 against PERV and found that SAMHD1 potently restricts its reverse transcription in human monocytes, monocyte-derived dendritic cells (MDDC), or macrophages (MDM) and in monocytic THP-1 cells. Degradation of SAMHD1 by SIVmac Vpx or CRISPR/Cas9 knock-out of SAMHD1 allowed for PERV reverse transcription. Addition of deoxynucleosides alleviated the SAMHD1-mediated restriction suggesting that SAMHD1-mediated degradation of dNTPs restricts PERV replication in these human immune cells. In conclusion, our findings highlight SAMHD1 as a potential barrier to PERV transmission from pig transplants to human recipients during xenotransplantation.

Generation of Human Stem Cell-Derived Pancreatic Organoids (POs) for Regenerative Medicine.

Insulin-dependent diabetes mellitus or type 1 diabetes mellitus (T1DM) is an auto-immune condition characterized by the loss of pancreatic β-cells. The curative approach for highly selected patients is the pancreas or the pancreatic islet transplantation. Nevertheless, these options are limited by a growing shortage of donor organs and by the requirement of immunosuppression.Xenotransplantation of porcine islets has been extensively investigated. Nevertheless, the strong xenoimmunity and the risk of transmission of porcine endogenous retroviruses, have limited their application in clinic. Generation of β-like cells from stem cells is one of the most promising strategies in regenerative medicine. Embryonic, and more recently, adult stem cells are currently the most promising cell sources exploited to generate functional β-cells in vitro. A number of studies demonstrated that stem cells could generate functional pancreatic organoids (POs), able to restore normoglycemia when implanted in different preclinical diabetic models. Nevertheless, a gradual loss of function and cell dead are commonly detected when POs are transplanted in immunocompetent animals. So far, the main issue to be solved is the post-transplanted islet loss, due to the host immune attack. To avoid this hurdle, nanotechnology has provided a number of polymers currently under investigation for islet micro and macro-encapsulation. These new approaches, besides conferring PO immune protection, are able to supply oxygen and nutrients and to preserve PO morphology and long-term viability.Herein, we summarize the current knowledge on bioengineered POs and the stem cell differentiation platforms. We also discuss the in vitro strategies used to generate functional POs, and the protocols currently used to confer immune-protection against the host immune attack (micro- and macro-encapsulation). In addition, the most relevant ongoing clinical trials, and the most relevant hurdles met to move towards clinical application are revised.

Will Genetic Engineering Carry Xenotransplantation of Pig Islets to the Clinic?

Porcine islets represent a potentially attractive beta-cell source for xenotransplantation into patients with type 1 diabetes, who are not eligible to islet allo-transplantation due to a lack of suitable human donor organs. Recent progress in genetic engineering/gene editing of donor pigs provides new opportunities to overcome rejection of xeno-islets, to improve their engraftment and insulin secretion capacity, and to reduce the risk for transmission of porcine endogenous retroviruses. This review summarizes the current issues and progress in islet xenotransplantation with special emphasis on genetically modified/gene edited donor pigs. Attempts to overcome acute rejection of xeno-islets, especially after intraportal transplantation into the liver, include the genetic elimination of specific carbohydrate antigens such as αGal, Neu5Gc, and Sd(a) for which humans and-in part-non-human primates have natural antibodies that bind to these targets leading to activation of complement and coagulation. A complementary approach is the expression of one or more human complement regulatory proteins (hCD46, hCD55, hCD59). Transgenic attempts to overcome cellular rejection of islet xenotransplants include the expression of proteins that inhibit co-stimulation of T cells. Expression of glucagon-like peptide-1 and M3 muscarinic receptors has been shown to increase the insulin secretion of virally transduced porcine islets in vitro and it will be interesting to see the effects of these modifications in transgenic pigs and islet products derived from them. Genome-wide inactivation of porcine endogenous retrovirus (PERV) integrants by mutating their pol genes using CRISPR/Cas9 is a recent approach to reduce the risk for PERV transmission by xeno-islets. Genetic engineering/gene editing of xeno-islet donor pigs facilitated major progress towards clinical islet xenotransplantation. The required set of genetic modifications will depend on the source of islets (fetal/neonatal vs. adult), the mode of delivery (encapsulated vs. free), and the transplantation site.

Assessment of porcine endogenous retrovirus transmission across an alginate barrier used for the encapsulation of porcine islets

Subcutaneous implantation of a macroencapsulated patch containing human allogenic islets has been successfully used to alleviate type 1 diabetes mellitus (T1DM) in a human recipient without the need for immunosuppression. The use of encapsulated porcine islets to treat T1DM has also been reported. Although no evidence of pathogen transfer using this technology has been reported to date, we deemed it appropriate to determine if the encapsulation technology would prevent the release of virus, in particular, the porcine endogenous retrovirus (PERV). HEK293 (human epithelial kidney) and swine testis (ST) cells were co-cultured with macroencapsulated pig islets embedded in an alginate patch, macroencapsulated PK15 (swine kidney epithelial) cells embedded in an alginate patch and free PK15 cells. Cells and supernatant were harvested at weekly time points from the cultures for up to 60days and screened for evidence of PERV release using qRT-PCR to detect PERV RNA and SG-PERT to detect reverse transcriptase (RT). No PERV virus, or evidence of PERV replication, was detected in the culture medium of HEK293 or pig cells cultured with encapsulated porcine islets. Increased PERV activity relative to the background was not detected in ST cells cultured with encapsulated PK15 cells. However, PERV was detected in 1 of the 3 experimental replicates of HEK293 cells cultured with encapsulated PK15 cells. Both HEK293 and ST cells cultured with free PK15 cells showed an increase in RT detection. With the exception of 1 replicate, there does not appear to be evidence of transmission of replication competent PERV from the encapsulated islet cells or the positive control PK15 cells across the alginate barrier. The detection of PERV would suggest the alginate barrier of this replicate may have become compromised, emphasizing the importance of quality control when producing encapsulated islet patches.

Human-APOBEC3G-dependent restriction of porcine endogenous retrovirus replication is mediated by cytidine deamination and inhibition of DNA strand transfer during reverse transcription.

Although human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G, hA3G)-mediated deamination is the major mechanism used to restrict the infectivity of a broad range of retroviruses, it is unclear whether porcine endogenous retrovirus (PERV) is affected by hA3G or porcine A3F (poA3F). To determine whether DNA deamination is required for hA3G- and poA3F-dependent inhibition of PERV transmission, we developed VSV-pseudotype PERV-B expressing hA3G, mutant hA3G-E67Q (encapsidation and RNA binding activity-deficient), mutant hA3G-E259Q (deaminase-deficient), or poA3F. hA3G-E67Q decreased virus infectivity by ~93% compared to the ~99% decrease of viral infectivity by wild-type hA3G, while hA3G-E259Q decreased the infectivity of PERV-B by ~35%. These data suggest that cytidine deamination activity is crucial for efficient restriction of PERV by hA3G, but cytidine deamination cannot fully explain the inactivation of PERV by hA3G. Furthermore, differential DNA denaturation PCR (3D-PCR) products from 293T cells infected with PERV-B expressing hA3G mutants were sequenced. G-to-A hypermutation was detected at a frequency of 4.1% for hA3G, 3.4% for hA3G-E67Q, and 4.7% for poA3F. These results also suggest that hA3G and poA3F inhibit PERV by a deamination-dependent mechanism. To examine the effect of hA3G on the production of PERV DNA, genomic DNA was extracted from 293T cells 12 h after infection with PERV expressing hA3G, and this DNA was used as template for real-time PCR. A 50% decrease in minus strand strong stop (-sss) DNA synthesis/transfer was observed in the presence of hA3G. Based on these results, we conclude that hA3G may restrict PERV by both deamination-dependent mechanisms and inhibition of DNA strand transfer during PERV reverse transcription.

Xenoislets: porcine pancreatic islets for the treatment of type I diabetes.

Porcine islets are being extensively investigated as alternative sources of insulin-secreting cells for transplantation in insulin-dependent diabetic patients. The present review focuses on recent advances in porcine islet transplantation with particular emphasis on new transgenic pig models, islet encapsulation, and biosafety considerations. Genetic modifications aimed to reduce islet cell immunogenicity, to prolong their survival, and to improve their secretory function have been reported. Micro- and macroencapsulation of porcine islets should allow their use in the clinic with no or minimal immunosuppression. The risk of porcine endogenous retrovirus transmission is being re-evaluated since no evidence for infection was found in several clinical and preclinical studies. Pig islet xenotransplantation is still a serious contestant in the race for novel treatments for type I diabetes. Adequate pathogen screening, animal selection, and the establishment of microbiological, genetic, and potency release quality controls should increase safety and efficacy of future porcine islets transplantation clinical trials.

Corneal Xenotransplantation From Pig to Rhesus Monkey: No Signs of Transmission of Endogenous Porcine Retroviruses

BackgroundXenotransplantations of pig corneas have become an attractive alternative to human corneas. Such xenotransplantations carry the danger, however, of transmission of porcine endogenous retroviruses (PERVs). Here, we investigated whether porcine corneas harbor viral DNA and RNA and whether transplantation to a nonhuman primate would lead to host PERV infection. MethodsMonkey vein endothelial cells (MVECs) were inoculated with porcine aortic endothelial cell (PAEC) supernatants, and DNA and total RNA of MVECs were tested for PERV by polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) assays. Corneas were harvested from Wuzhishan miniature pigs, and the presence of PERV proviral DNA and RNA was analyzed by PCR and RT-PCR, respectively. Fresh or dehydrated corneas were then transplanted to rhesus monkeys, and PERV proviral DNA and RNA were analyzed in host peripheral blood lymphocytes at 6 and 24 months. Furthermore, the presence of PERV sequences was analyzed in the transplant at 24 months. ResultsPCR analysis showed PERV transfection from PAECs to MVECs in vitro. PCR and RT-PCR gave positive signals for PERV subtypes A and B, but not PERV-C, regardless of how the corneas were prepared. No evidence was found for PERV transmission to the host, and the transplant had lost its viral signal at the end of the 24-month period. ConclusionsRegardless of cornea preparation and storage, PERV transmission from pig to host could not be detected, despite that the transplant was initially PERV-positive. The use of the Wuzhishan miniature pig as the donor may be advantageous because it lacks PERV-C and hence potentially infectious A/C recombinants.

Pig islet xenotransplantation

The current article reviews the rationale, sources and preparation of pig islets for xenotransplantation, and presents current progress in solving the problems associated with establishing pig islet transplant as a clinical treatment for type 1 diabetes. Islet transplantation is an effective treatment option for type 1 diabetes, but the available supply of human pancreases is insufficient to meet the need and demand for obtaining islets. Pig islets provide a readily available source for islet transplantation, with trials in non-human primates demonstrating their potential to reverse diabetes. The risk of zoonosis can be reduced by designated pathogen-free breeding of the donor pigs, but porcine endogenous retroviruses (PERVs) that are integrated into the genome of all pigs are especially difficult to eliminate. However, clinical trials have demonstrated an absence of PERV transmission with a significant reduction in the number of severe hypoglycemic episodes and up to 30% reduction in exogenous insulin doses. A number of methods such as production of various transgenic pigs to better xenotransplantation efficiency and the encapsulation of islets to isolate them from the host immune system are currently being tested to overcome the xenograft immune rejection. Furthermore, ongoing research is also shedding light on factors such as the age and breed of the donor pig to determine the optimal islet quantity and function.

Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9.

Xenotransplantation is a promising strategy to alleviate the shortage of organs for human transplantation. In addition to the concerns about pig-to-human immunological compatibility, the risk of cross-species transmission of porcine endogenous retroviruses (PERVs) has impeded the clinical application of this approach. We previously demonstrated the feasibility of inactivating PERV activity in an immortalized pig cell line. We now confirm that PERVs infect human cells, and we observe the horizontal transfer of PERVs among human cells. Using CRISPR-Cas9, we inactivated all of the PERVs in a porcine primary cell line and generated PERV-inactivated pigs via somatic cell nuclear transfer. Our study highlights the value of PERV inactivation to prevent cross-species viral transmission and demonstrates the successful production of PERV-inactivated animals to address the safety concern in clinical xenotransplantation.

Can Antiretroviral Drugs Be Used to Treat Porcine Endogenous Retrovirus (PERV) Infection after Xenotransplantation?

Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pigs; they are released as infectious particles, and under certain conditions they can infect human cells. Therefore, they represent a risk when pigs are used as sources of cells, tissues, or organs for xenotransplantation. Xenotransplantation is under development due to the increasing shortage of human transplants. Whereas most porcine microorganisms which may be able to induce a disease (zoonosis) in the transplant recipient can be eliminated, this is not possible in the case of PERVs. Antiretroviral drugs which had been developed for the treatment of human immunodeficiency virus-1 (HIV-1) infections have been tested in vitro for their efficacy in inhibiting PERV replication. Inhibitors of the viral reverse transcriptase and of the integrase have been found effective. The most effective inhibitor of the reverse transcriptase was azidothymidine (AZT); the integrase inhibitors were the most potent inhibitors of PERV. Although in the past PERV transmission has not been observed after experimental or clinical xenotransplantation of pig cells or organs, and although PERVs may one day be inactivated in pigs by genome editing using CRISPR/Cas, knowing which antiretroviral drugs can effectively restrict PERV infection will still be important.

Long-term safety from transmission of porcine endogenous retrovirus after pig-to-non-human primate corneal transplantation.

The risk of xenozoonosis mainly by porcine endogenous retrovirus (PERV) has been considered as one of the main hurdles in xenotransplantation and therefore should be elucidated prior to the clinical use of porcine corneal grafts. Accordingly, an investigation was performed to analyze the infectivity of PERVs from porcine keratocytes to human cells, and the long-term risk of transmission of PERVs was determined using pig-to-non-human primate (NHP) corneal transplantation models. The infectivity of PERVs from the SNU miniature pig keratocytes was investigated by coculture with a human embryonic kidney cell line. Twenty-two rhesus macaques underwent xenocorneal transplantation as follows: (i) group 1 (n=4): anterior lamellar keratoplasty (LKP) with freshly preserved porcine corneas, (ii) group 2 (n=5): anterior LKP with decellularized porcine corneas followed by penetrating keratoplasty (PKP) with allografts, (iii) group 3 (n=3): PKP under steroid-based immunosuppression, (iv) group 4 (n=4): PKP under anti-CD154 antibody-based immunosuppression, (v) group 5 (n=4): deep anterior LKP with freshly preserved porcine corneas under anti-CD40 antibody-based immunosuppression, and (vi) group 6 (n=2): PKP under anti-CD40 antibody-based immunosuppression. Postoperative blood samples were serially collected, and tissue samples were obtained from thirteen different organs at the end of each experiment. The existence of PERV DNA and RNA was investigated using PCR and RT-PCR. Using two independent in vitro infectivity tests, neither PERV pol nor pig mitochondrial cytochrome oxidase II was detected after 41 and 92days of coculture, respectively. After xenocorneal transplantation, a total of 257 serial peripheral blood mononuclear cell samples, 34 serial plasma samples, and 282 tissue samples were obtained from the NHP recipients up to 1176days post-transplantation. No PERV transmission was evident in any samples. Within the limits of this study, there is no evidence to support any risk of PERV transmission from porcine corneal tissues to NHP recipients, despite the existence of PERV-expressing cells in porcine corneas.

The Resurgence of Xenotransplantation.

There has been an upsurge of interest in xenotransplantation in recent years. This resurgence can attributed to a combination of factors. First, there has been a dramatic improvement in efficacy in several preclinical models, with maximum xenograft survival times increasing to 950 days for islets, 945 days for hearts, and 310 days for kidneys. Second, the rapid development of genome editing technology (particularly the advent of clustered regularly interspaced short palindromic repeats/Cas9) has revolutionized the capacity to generate new donor pigs with multiple protective genetic modifications; what once took many years to achieve can now be performed in months, with much greater precision and scope. Third, the specter of porcine endogenous retrovirus (PERV) has receded significantly. There has been no evidence of PERV transmission in clinical trials and preclinical models, and improved screening methods and new options for the treatment or even elimination of PERV are now available. Balancing these positive developments are several remaining challenges, notably the heavy and often clinically inapplicable immunosuppression required to prevent xenograft rejection. Nonetheless, the potential for xenotransplantation as a solution to the shortage of human organs and tissues for transplantation continues to grow.

Recent Progress in Xenotransplantation, with Emphasis on Virological Safety.

Xenotransplantation is a new technology that may help to overcome the shortage of human tissues and organs available for the treatment of tissue and organ failure. Remarkable progress has recently been made in this field. First, understanding of the mechanisms of immunological rejection, mainly of the hyperacute rejection, allowed generating numerous genetically modified pigs to overcome rejection. Second, based on these genetically modified animals and new immunosuppression regimens, long-term survival of non-human primate recipients of heart, kidney, and islet cell cells has been reported. And third, potential zoonotic microorganisms have been identified in pigs and sensitive methods to detect them have been generated. In 2 clinical trials treating diabetic patients with porcine islet cells, no porcine microorganisms were transmitted to human recipients. Furthermore, strategies to eliminate potentially zoonotic microorganisms from donor pigs in order to prevent transmission to the recipients have been developed, including designated pathogen-free (DPF) breeding. In addition, strategies to prevent transmission of porcine endogenous retroviruses (PERVs) have been developed, including a knockout of all proviruses in the pig genome by gene editing. PERVs are integrated in the genome of all pigs and therefore they cannot be eliminated by DPF breeding. Since they are able to infect human cells, they represent a special risk in xenotransplantation. Despite the achievements, some problems remain: numerous genetically multi-modified pigs have been generated without fully evaluating their advantage, and microbiological screening of pigs to be used for transplantations and elimination of pathogenic microorganisms from the donor pigs are still not satisfactory.