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For many patients with terminal/advanced cardiac failure, heart transplantation is the most effective, durable treatment option, and offers the best prospects for a high quality of life. The number of potentially life-saving donated human organs is far fewer than the population who could benefit from a new heart, resulting in increasing numbers of patients awaiting replacement of their failing heart, high waitlist mortality, and frequent reliance on interim mechanical support for many of those deemed among the best candidates but who are deteriorating as they wait. Currently, mechanical assist devices supporting left ventricular or biventricular heart function are the only alternative to heart transplant that is in clinical use. Unfortunately, the complication rate with mechanical assistance remains high despite advances in device design and patient selection and management, and the quality of life of the patients even with good outcomes is only moderately improved. Cardiac xenotransplantation from genetically multi-modified (GM) organ-source pigs is an emerging new option as demonstrated by the consistent long-term success of heterotopic (non-life-supporting) abdominal and life-supporting orthotopic porcine heart transplantation in baboons, and by a recent ‘compassionate use’ transplant of the heart from a GM pig with 10 modifications into a terminally ill patient who survived for 2 months. In this review, we discuss pig heart xenotransplantation as a concept, including pathobiological aspects related to immune rejection, coagulation dysregulation, and detrimental overgrowth of the heart, as well as GM strategies in pigs to prevent or minimize these problems. Additional topics discussed include relevant results of heterotopic and orthotopic heart transplantation experiments in the pig-to-baboon model, microbiological and virologic safety concepts, and efficacy requirements for initiating formal clinical trials. An adequate regulatory and ethical framework as well as stringent criteria for the selection of patients will be critical for the safe clinical development of cardiac xenotransplantation, which we expect will be clinically tested during the next few years.

Yucatan SLA D/D haplotype miniature swine are candidates as organ donors for xenotransplantation. A full-length replication-competent PERV-C provirus was characterized. The provirus was chromosomally mapped in the pig genome. In vitro , the virus showed increased infectivity compared to other functional PERV-C isolates. Data may be used for targeted knockout to generate PERV-C free founder animals.

INTRODUCTION: Porcine endogenous retroviruses (PERVs) are an integral part of the pig genome with infectious potential, as shown in vitro. HYPOTHESIS/GAP STATEMENT: In view of nonclinical and clinical xenotransplantation, data are essential that give an insight into viral pathogenicity. This includes PERV's environmental stability and environmental risk.AIM: We analyzed two ecotropic PERV-C (PERV-C[1312] and -[5683]), monitoring cell-free culture supernatants of infected ST-IOWA cells at various time intervals at room temperature (22°C +/−1°C). The virus was stored in the presence or absence of sterile wood litter, as used for large animal husbandry. This approach was set to determine the environmental stability of exogenous PERV-C at defined conditions for the first time. METHODOLOGY: Reverse transcriptase (RT) activity and viral RNA were monitored for up to 57 days and remaining infectivity of supernatant without wood litter was tested from day 7 onwards on naïve ST-IOWA cells. RESULTS: Results show that viral RNA decreases but remains detectable over the whole observation period, whereas RT activity showed 83%–96% reduction from day 7 on. This effect was stronger in the presence of wood litter and fresh harvested virus was more stable than frozen virus stocks. Even under these optimal conditions, no infectivity was shown for viral RNA-positive and RT-reduced supernatant harvested at day 7. CONCLUSION: The results confirm that PERV-C is less stable and the reduction of RT activity is accompanied by reduced infectivity, independently of existing viral RNA. The combination of both RT and viral RNA measurement is a suitable method to differentiate infectious PERV-C.

Background The lack of human donors for allotransplantation forces the development of other strategies to circumvent the existing organ shortage documented on the waiting lists. Here, xenotransplantation offers a suitable option since the genetic modification of animals has become an established method that allows the generation of animals as donors of cells, tissues, and organs with reduced antigenicity. Methods Focus is given on the generation of decellularized matrix scaffolds, for example, for valve transplantation and/or repair, that have the potential being fully assimilated by the recipient as they are no longer a mechanical implant with risk of calcification and related failure. Results This new class of products is transplants that will be regulated either as medical devices or as cell-based medicinal products, that is, advanced therapy medicinal products, according to the regulations in the European Union. Conclusions In this review, we compile relevant regulatory aspects and point out the possibilities of how these products for human use may be regulated in the future.

Background The demand for donated human hearts far exceeds the number available. Xenotransplantation of genetically modified porcine organs provides an alternative. In 2000, an Advisory Board of the International Society for Heart and Lung Transplantation set the benchmark for commencing clinical cardiac xenotransplantation as consistent 60% survival of non-human primates after life-supporting porcine heart transplantations. Recently, we reported the stepwise optimization of pig-to-baboon orthotopic cardiac xenotransplantation finally resulting in consistent success, with 4 recipients surviving 90 (n = 2), 182, and 195 days. Here, we report on 4 additional recipients, supporting the efficacy of our procedure. Results The first 2 additional recipients succumbed to porcine cytomegalovirus (PCMV) infections on Days 15 and 27, respectively. In 2 further experiments, PCMV infections were successfully avoided, and 3-months survival was achieved. Throughout all the long-term experiments, heart, liver, and renal functions remained within normal ranges. Post-mortem cardiac diameters were slightly increased when compared with that at the time of transplantation but with no detrimental effect. There were no signs of thrombotic microangiopathy. The current regimen enabled the prolonged survival and function of orthotopic cardiac xenografts in altogether 6 of 8 baboons, of which 4 were now added. These results exceed the threshold set by the Advisory Board of the International Society for Heart and Lung Transplantation. Conclusions The results of our current and previous experimental cardiac xenotransplantations together fulfill for the first time the pre-clinical efficacy suggestions. PCMV-positive donor animals must be avoided.

BACKGROUND: Decellularized human pulmonary heart valve (dhHV) scaffolds have been shown to be the gold standard especially for younger, adolescent patients. However, human heart valves are limited in availability. Xenogeneic decellularized pig heart valves (dpHV) may serve as alternative. METHODS: The efficacy of DNA reduction processes upon decellularization of heart valves from German Landrace pigs was analyzed by measurements of remaining nucleic acids including proviral porcine endogenous retrovirus (PERV) sequences. Porcine pulmonary heart valves (pPHV) were decellularized by three different protocols and further treated with DNaseI or Benzonase, at varying incubation times. DNA isolated from valve associated muscle (m), valve cusp (c), and pulmonary artery (pa) was monitored by PCR and qRT-PCR using GAPDH and the PERV polymerase (pol) for read-out. RESULTS: Decellularization of pPHV led to a significant reduction of DNA (>99%) which could be further significantly increased for (m) and (pa) by nuclease treatment, reducing proviral PERV pol from approximately 5 × 107 to 5 × 103 copies/mg in nuclease treated tissues. CONCLUSIONS: Both nucleases demonstrated comparable activities. But DNaseI revealed to be less consistent for PERV, especially at muscular tissue. Noteworthy, remaining proviral sequences are still detectable by PCR; however, due to the absence of the cellular replication machinery the production of infectious particles is not expected. Decellularization and nuclease treatment of pPHV is an efficient procedure to reduce the DNA content including PERV, thus represents a valuable option to increase virus safety independently from the source animal background.

Xenotransplantation of porcine cells, tissues, and organs may be associated with the transmission of porcine microorganisms to the human recipient. A previous, 2009, version of this consensus statement focused on strategies to prevent transmission of porcine endogenous retroviruses (PERVs). This version addresses potential transmission of all porcine microorganisms including monitoring of the recipient and provides suggested approaches to the monitoring and prevention of disease transmission. Prior analyses assumed that most microorganisms other than the endogenous retroviruses could be eliminated from donor animals under appropriate conditions which have been called "designated pathogen-free" (DPF) source animal production. PERVs integrated as proviruses in the genome of all pigs cannot be eliminated in that manner and represent a unique risk. Certain microorganisms are by nature difficult to eliminate even under DPF conditions; any such clinically relevant microorganisms should be included in pig screening programs. With the use of porcine islets in clinical trials, special consideration has to be given to the presence of microorganisms in the isolated islet tissue to be used and also to the potential use of encapsulation. It is proposed that microorganisms absent in the donor animals by sensitive microbiological examination do not need to be monitored in the transplant recipient; this will reduce costs and screening requirements. Valid detection assays for donor and manufacturing-derived microorganisms must be established. Special consideration is needed to preempt potential unknown pathogens which may pose a risk to the recipient. This statement summarizes the main achievements in the field since 2009 and focus on issues and solutions with microorganisms other than PERV.

Islet xenotransplantation represents an attractive solution to overcome the shortage of human islets for use in type 1 diabetes. The wide-scale application of clinical islet xenotransplantation, however, requires that such a procedure takes place in a specifically and tightly regulated environment. With a view to promoting the safe application of clinical islet xenotransplantation, a few years ago the International Xenotransplantation Association (IXA) published a Consensus Statement that outlined the key ethical and regulatory requirements to be satisfied before the initiation of xenotransplantation studies in diabetic patients. This earlier IXA Statement also documented a disparate regulatory landscape among different geographical areas. This situation clearly fell short of the 2004 World Health Assembly Resolution WHA57.18 that urged Member States "to cooperate in the formulation of recommendations and guidelines to harmonize global practices" to ensure the highest ethical and regulatory standards on a global scale. In this new IXA report, IXA members who are active in xenotransplantation research in their respective geographic areas herewith briefly describe changes in the regulatory frameworks that have taken place in the intervening period in the various geographic areas or countries. The key reassuring take-home message of the present report is that many countries have embraced the encouragement of the WHO to harmonize the procedures in a more global scale. Indeed, important regulatory changes have taken place or are in progress in several geographic areas that include Europe, Korea, Japan, and China. Such significant regulatory changes encompass the most diverse facets of the clinical application of xenotransplantation and comprise ethical aspects, source animals and product specifications, study supervision, sample archiving, patient follow-up and even insurance coverage in some legislations. All these measures are expected to provide a better care and protection of recipients of xenotransplants but also a higher safety profile to xenotransplantation procedures with an ultimate net gain in terms of international public health.

Background Porcine induced pluripotent stem cells (piPSCs) offer an alternative strategy in xenotransplantation (XTx). As human endogenous retroviruses (HERV), particularly HERV‐K, are highly expressed in natural human stem cells, we compared the expression of porcine endogenous retroviruses (PERV) and retrotransposon LINE‐1 (L1) open reading frames 1 and 2 (pORF1 and pORF2) in different piPSC‐like cell lines with their progenitors (porcine fetal fibroblasts, pFF). Methods Cells reprogrammed via Sleeping Beauty‐transposed transcription factors were cultured and analyzed on a custom‐designed microarray representing the reference pig genome. Data were complemented by qRT‐PCR and reverse transcriptase (RT) assay. Results The expression profiles revealed that 8515 of 26 967 targets were differentially expressed. A total of 4443 targets showed log2 expression ratio >1, and 4072 targets showed log2 expression ratio less than −1 with 0.05 P‐value threshold. Approximately ten percent of the targets showed highly significant expression ratios with log2 ≥4 or ≤−4. Besides this general switch in cellular gene expression that was accompanied by an altered morphology, expression of both PERV and L1 pORF1/pORF2 was significantly enhanced. piPSC‐like cells revealed a 10‐fold to 100‐fold higher transcription of the viral PERV‐A and PERV‐B envelope genes (env), viral protease/polymerase (prt/pol), and L1 elements. No functional retrovirus could be detected under these conditions. Conclusion Epigenetic reprogramming has functional impact on retrotransposons. Thus, the induction of pig‐derived pluripotent cells influences their PERV expression profile. Data emphasize the necessity to focus on animals, which show non‐functional endogenous viral background to ensure virological safety.

Angesichts der bestehenden Knappheit an humanen Spenderorganen und -geweben kann die Entwicklung xenogener Zelltherapeutika (xZT) eine adaquate Behandlungsalternative sein. Insbesondere porcine Pankreasinseln und -inselzellen haben bereits zum jetzigen Zeitpunkt Eingang in die experimentelle Therapie von Typ 1 Diabetes mellitus (T1DM)-Patienten gefunden. Dabei stellen xZT sowohl aus medizinischer als auch aus ethischer und regulatorischer Sicht eine Herausforderung dar. So ist infolge der speziesubergreifenden Transplantation (Xenotransplantation) neben dem Risiko der immunologischen Abstosung auch das Infektionsrisiko durch Ubertragung von mikrobiellen und viralen Erregern zu berucksichtigen. Letzteres ist bidirektional und betrifft sowohl die Transmission vom Transplantat auf den Empfanger als auch die Transmission vom Empfanger auf das Transplantat. Die Uberschreitung der Speziesbarriere erfordert eine kritische Nutzen-Risiko-Bewertung und nach Abschluss der Behandlung eine sorgfaltige und langfristige Uberwachung der Patienten. Die internationalen rechtlichen und regulatorischen Anforderungen an xZT basieren u. a. auf den von der Weltgesundheitsorganisation (WHO) 2008 im Changsha Kommunique formulierten Kriterien, die in Europa nach Inkraftsetzen der EU-Verordnung uber Arzneimittel fur neuartige Therapien (ATMP) im Leitfaden fur xenogene Zelltherapeutika seitens der Europaischen Arzneimittelbehorde EMA mit abgebildet sind.

Xenotransplantation of porcine organs, tissues, and cells inherits a risk for xenozoonotic infections. Viable tissues and cells intended for transplantation have to be considered as potentially contaminated non-sterile products. The demands on microbial testing, based on the regulatory requirements, are often challenging due to a restricted shelf life or the complexity of the product itself. In Europe, the regulatory framework for xenogeneic cell therapy is based on the advanced therapy medicinal products (ATMP) regulation (2007), the EMA CHMP Guideline on xenogeneic cell-based medicinal products (2009), as well as the WHO and Council of Europe recommendations. In the USA, FDA guidance for industry (2003) regulates the use of xenotransplants. To comply with the regulations, validated test methods need to be established that reveal the microbial status of a transplant within its given shelf life, complemented by strictly defined action alert limits and supported by breeding in specific pathogen-free (SPF) facilities. In this review, we focus on assays for the detection of the porcine endogenous retroviruses PERV-A/-B/-C, which exhibit highly polymorphic proviral loci in pig genomes. PERVs are transmitted vertically and cannot be completely eliminated by breeding or gene knock out technology. PERVs entail a public health concern that will persist even if no evidence of PERV infection of xenotransplant recipients in vivo has been revealed yet. Nevertheless, infectious risks must be minimized by full assessment of pigs as donors by combining different molecular screening assays for sensitive and specific detection as well as a functional analysis of the infectivity of PERV including an adequate monitoring of recipients.

Background Porcine endogenous retroviruses (PERV) pose a zoonotic risk potential in pig-to-human xenotransplantation given that PERV capacity to infect different human cell lines in vitro has been clearly shown in the past. However, PERV infectious potential for human peripheral blood mononuclear cells (huPBMC) has been also demonstrated, albeit with controversial results. As productive PERV infection of huPBMC involves immune suppression that may attract opportunistic pathogens as shown for other retroviruses, it is crucial to ascertain unequivocally huPBMC susceptibility for PERV. To address this question, we first investigated in vitro infectivity of PERV for huPBMC using supernatants containing highly infectious PERV-A/C. Second, huPBMC were cocultivated with PERV-A/C producer cells to come a step closer to the in vivo situation of xenotransplantation. In addition, cocultivation of huPBMC with porcine PBMC (poPBMC) isolated from German landrace pigs was performed to distinguish PERV replication competence when they were constitutively produced by immortalized cells or by primary poPBMC. Methods Supernatants containing recombinant highly infectious PERV-A/C were used to infect PHA-activated huPBMC in the presence or absence of polybrene. Next, PERV-producing cell lines such as human 293/5° and primary mitogenically activated poPBMC of three German landrace pigs were cocultivated with huPBMC as well as with susceptible human and porcine cell lines as controls. PERV infection was monitored by using three test approaches. The presence of provirus DNA in putatively infected cells was detected via sensitive nested PCR. Viral expression was determined by screening for the activity of gammaretroviral reverse transcriptase (RT) in cell-free supernatants of infected cells. Virus release was monitored by counting the number of packaged RNA particles in supernatants via PERV-specific quantitative one-step real-time reverse transcriptase PCR. Results Porcine endogenous retroviruses-A/C in supernatants of human producer 293/5° cells was not able to infect huPBMC. Neither RT activity nor PERV copies were detected. Even provirus could not be detected displaying the inability of PERV-A/C to induce a productive infection in huPBMC. In cocultivation experiments only non-productive infection of huPBMC with PERV derived from 293/5° cell line and from PHA-activated poPBMC was observed by detection of provirus DNA in infected cells. Conclusion Recombinant PERV-A/C in supernatants of producer cells failed to infect huPBMC, whereas coculture experiments with producer cell lines lead to non-productive infection of huPBMC. PERV in supernatants seem to have not sufficient infectious potential for huPBMC. However, extensive PERV exposure to huPBMC via cocultivation enabled at least virus cell entry as provirus was detected by nested PCR. Furthermore, results presented support previous data showing German landrace pigs as low producers with negligible infectious potential due to the absence of replication-competent PERV in the genome. The low PERV expression profile and the lack of significant replication competence of German landrace pigs raise hope for considering these animals as putative donor animals in future pig-to-human xenotransplantation. Nonetheless, data imply that PERV still represent a virological risk in the course of xenotransplantation, as the presence of PERV provirus in host cells may lead to a provirus integration resulting in insertional mutagenesis and chromosomal rearrangements.

After feedback from the working parties, the final session focused on drafting proposed revisions of the WHO documents, and resulted in the formulation of the draft “Third WHO Global Consultation on Regulatory Requirements for Xenotransplantation Clinical Trials, The 2018 Changsha Communiqué.” This draft was submitted to WHO in February 2019 for WHO and World Health Assembly consideration. If approved, the 2018 Changsha Communiqué will then be posted on the websites of WHO, IXA, and TTS, and published in Xenotransplantation. This report includes summaries of the various sessions, followed by the abstracts of invited speakers from the update sessions.

Seit Inkrafttreten des Gewebegesetzes in Deutschland am 1. August 2007 ist jede Gewebeeinrichtung gemas § 8d Abs. 3 Transplantationsgesetz (TPG) dazu verpflichtet, eine Dokumentation uber ihre Tatigkeit zu fuhren und dem Paul-Ehrlich-Institut einmal jahrlich bis zum 1. Marz des Folgejahres einen Bericht zu ubermitteln. Hierin sollen Angaben zur Art und Menge der entnommenen, aufbereiteten, be- oder verarbeiteten, aufbewahrten, abgegebenen oder anderweitig verwendeten, eingefuhrten und ausgefuhrten Gewebe gemacht werden. Die TPG-Meldung soll auf einem vom Paul-Ehrlich-Institut im Internet veroffentlichen Meldebogen erfolgen. Dem vorliegenden Gesamtbericht nach § 8d Abs. 3 TPG liegen Daten zugrunde, die sich auf die Meldejahre 2009 bis 2011 beziehen. Sechs Jahre nach Einfuhrung der TPG-Meldepflicht fur Gewebeeinrichtungen ist die Zahl der dem Paul-Ehrlich-Institut bekannten meldepflichtigen Gewebeeinrichtungen von 349 fur das Jahr 2007 auf 949 fur das Jahr 2011 angestiegen. Nach kontinuierlicher Optimierung der Meldebogen inklusive gewebespezifischer Glossare sind die gemeldeten Daten fur die meisten Gewebe und Gewebezubereitungen mittlerweile relativ schlussig.

Das Gewebegesetz ist in Deutschland am 1. August 2007 in Kraft getreten. In diesem Artikelgesetz wurden die EU-Richtlinien zur Qualitat und Sicherheit von menschlichen Geweben und Zellen in das Transplantationsgesetz (TPG) und Arzneimittelgesetz implementiert. Seitdem ist jede Gewebeeinrichtung verpflichtet, eine Dokumentation uber ihre Tatigkeit zu fuhren und dem Paul-Ehrlich-Institut (PEI) jahrlich einen Bericht zu ubermitteln. Hierin sollen Angaben zur Art und Menge der entnommenen, aufbereiteten, be- oder verarbeiteten, aufbewahrten, abgegebenen oder anderweitig verwendeten, eingefuhrten und ausgefuhrten Gewebe gemacht werden. Das PEI hat hierzu einen TPG-Meldebogen im Bundesanzeiger und im Internet veroffentlicht. Die von den Gewebeeinrichtungen ubermittelten Angaben wurden anonymisiert in einem Gesamtbericht zusammengestellt. Die Auswertung zeigt, dass viele Daten in sich nicht schlussig sind, welches verschiedene Grunde haben kann. Um zukunftig eine hohere Konsistenz in Bezug auf die gemeldeten Daten zu erreichen, werden die Erlauterungen zum Ausfullen des Meldebogens erganzt. Die erhobenen Daten sind bisher nicht geeignet, zuverlassige Aussagen uber die Verfugbarkeit von Gewebe/Gewebezubereitungen in Deutschland zu treffen, sondern dienen als Anhaltspunkte.

Xenotransplantation of porcine cells, tissues, and organs shows promise to surmount the shortage of human donor materials. Among the barriers to pig-to-human xenotransplantation are porcine endogenous retroviruses (PERV) since functional representatives of the two polytropic classes, PERV-A and PERV-B, are able to infect human embryonic kidney cells in vitro , suggesting that a xenozoonosis in vivo could occur. To assess the capacity of human and porcine cells to counteract PERV infections, we analyzed human and porcine APOBEC3 (A3) proteins. This multigene family of cytidine deaminases contributes to the cellular intrinsic immunity and act as potent inhibitors of retroviruses and retrotransposons. Our data show that the porcine A3 gene locus on chromosome 5 consists of the two single-domain genes A3Z2 and A3Z3 . The evolutionary relationships of the A3Z3 genes reflect the evolutionary history of mammals. The two A3 genes encode at least four different mRNAs: A3Z2, A3Z3, A3Z2-Z3, and A3Z2-Z3 splice variant A (SVA). Porcine and human A3s have been tested toward their antiretroviral activity against PERV and murine leukemia virus (MuLV) using novel single-round reporter viruses. The porcine A3Z2, A3Z3 and A3Z2-Z3 were packaged into PERV particles and inhibited PERV replication in a dose-dependent manner. The antiretroviral effect correlated with editing by the porcine A3s with a trinucleotide preference for 5′ TGC for A3Z2 and A3Z2-Z3 and 5′ CAC for A3Z3. These results strongly imply that human and porcine A3s could inhibit PERV replication in vivo , thereby reducing the risk of infection of human cells by PERV in the context of pig-to-human xenotransplantation.

The clinical application of xenotransplantation poses immunologic, ethical, and microbiologic challenges. Significant progress has been made in the investigation of each of these areas. Among concerns regarding infectious risks for human xenograft recipients is the identification in swine of infectious agents including porcine endogenous retroviruses (PERV) that are capable of replication in some human cell lines. PERV replication has, however, been difficult to demonstrate in primate-derived cell lines and in preclinical studies of non-human primates receiving porcine xenografts. Endogenous 'retroviral restriction factors' are intracellular proteins and components of the innate immune system that act at various steps in retroviral replication. Recent studies suggest that some of these factors may have applications in the management of endogenous retroviruses in xenotransplantation. The risks of PERV infection and the potential role of retroviral restriction factors in xenotransplantation are reviewed in detail.

Background For xenotransplantation, strategies to prevent transmission of microorganisms from the source animal to the human recipient must be closely coordinated since tissues and organs are classified as non-sterile. Strategies for international cooperation and coordination of xenogeneic infection / disease surveillance and response are available. Methods The regulatory frameworks and criteria on microbial safety as published by World Health Organization (WHO), European Pharmacopoeia (Ph. Eur.), European Medicines Agency (EMA) as well as U.S. Department of Health and Human Services (DHHS), Food and Drug Administration (FDA) and Center for Biologics Evaluation and Research (CBER), are outlined. Results Different sources of microbial germs are considered including potential infectious agents. Monitoring of livestock and testing of xenografts is accompanied by positive and negative controls to detect and to exclude tissue specific microorganisms such as bacteria. Conclusions The criteria of microbial status to be considered for xenotransplants are summarized.

Of all human endogenous retroviruses known today, HERV-K is the only one that has been shown to produce viral particles. While the first of the approximately 30 HERV-K sequences integrated into the human genome more than 40 million years ago, evidence is accumulating that HERV-K was active more recently, provirus HERV-K113 being the youngest sequence found. However, it is unclear which HERV-K sequences code for the viral particles that are produced by human germ-cell tumours or melanomas. Here, we show that the provirus HERV-K113, cloned into a baculovirus expression vector, is capable of producing intact particles of retroviral morphology, exhibiting the typical structure of those particles that were characterized in cell lines derived from human germ-cell tumours. Thus, the HERV-K113 sequence is a candidate for particle production in vivo and for an active human endogenous retrovirus of today.

Am 1. August 2007 trat in Deutschland das Gewebegesetz in Kraft, welches unter anderem Regelungen hinsichtlich der Qualitat und Sicherheit von Gewebezubereitungen (GWZ) trifft. Klassische GWZ sind Arzneimittel fur die Anwendung am Menschen, a) die menschliche Gewebe und Zellen enthalten oder ausgehend von diesen hergestellt werden, b) deren wesentliche Be- oder Verarbeitungsverfahren in der EU hinreichend bekannt bzw. neu, aber mit bekannten Verfahren vergleichbar sind, und c) deren Wirkungen und Nebenwirkungen aus dem wissenschaftlichen Erkenntnismaterial ersichtlich sind. Dazu gehoren z.B. Herzklappen, Corneae, muskuloskelettale GWZ, Gefase oder Stammzellen aus Knochenmark. Wenn bei einer GWZ die oben genannten Voraussetzungen erfullt sind und diese nicht mit ionisierenden Strahlen behandelt wird, ist fur das Inverkehrbringen eine Genehmigung gemas § 21a Abs. 1 Arzneimittelgesetz (AMG) durch das Paul-Ehrlich-Institut erforderlich. Zur Erleichterung der Antragstellung fur die Genehmigung stehen Formulare auf der Internetseite des Paul-Ehrlich-Instituts zur Verfugung. Schwerwiegende unerwunschte Reaktionen und schwerwiegende Zwischenfalle gemas § 63c AMG mussen von Gewebeeinrichtungen unverzuglich an die zustandige Bundesbehorde gemeldet werden. Des Weiteren besteht fur Gewebeeinrichtungen eine Meldepflicht gemas § 8d Abs. 3 Transplantationsgesetz (TPG) zur Erfassung der verarbeiteten Gewebe, die in Form eines jahrlichen Berichts bis zum 1. Marz des folgenden Jahres an das Paul-Ehrlich-Institut erfolgt.

In view of the existing shortage of human donor organs and tissues, xenogeneic cell therapeutics (xCT) offer an alternative for adequate treatment. In particular, porcine pancreatic islets and islet cells have already entered the field of experimental therapy for type-1 diabetes mellitus (T1DM) patients. Thereby, xCT depict challenging products with a glance on medical, ethical, and regulatory questions. With cross-species transplantation (xenotransplantation), the risk of immunological graft rejection as well as the risk of infectious transmission of microbial and viral pathogens must be considered. This includes the bidirectional transmission of microorganisms from graft to host as well as from host to graft. Crossing the border of species requires a critical risk-benefit evaluation as well as a thorough longtime surveillance of transplant recipients after treatment. The international legal and regulatory requirements for xCT are inter alia based on the World Health Organization criteria summarized in the Changsha Communiqué (2008). In the European Union, they were reflected by the European Medicines Agency (EMA) Guideline on Xenogeneic Cell-based Medicinal Products following the implementation of the Regulation on Advanced Therapies (ATMP). On the basis of this regulation, the first non-clinical and clinical experiences were obtained for porcine islets. The results suggest that supportive treatment of T1DM risk patients with xCT may be an alternative to established allogeneic organ transplantation in the future.

Different Suiformes with increasing phylogenetic distance to the common pig ( Sus scrofa ) were assayed for the presence of porcine endogenous retroviruses (PERV) in general ( pol gene), while the distribution of long terminal repeat (LTR) types (with or without repeats in U3) and env genes (classes A, B, and C) were determined in detail. PERV was not detectable in the most distantly related species, while classes PERV-A and PERV-B are present in Suiformes originating in the Pliocene epoch, and class PERV-C was detectable only in S. scrofa and in closely related species originating in the Holocene epoch. This distribution pattern of PERV classes is in line with our previous study on the age of PERV (45) and suggests an African origin of about 7.5 million years ago (MYA) and a gradual spread of PERV through the Suiformes . It seems likely that PERV-C originated more recently (1.5 to 3.5 MYA) by recombination with a homologue of unknown descent, while the origin of the repeatless LTR was a separate event approximately 3.5 MYA.

The study of porcine endogenous retroviruses (PERVs) becomes increasingly important due to the potential use of pig cells, tissues, and organs as a source for xenogenic cell therapy and xenotransplantation into humans. Consequently, we have constructed a plasmid that induces in bacteria the synthesis of a soluble and highly active reverse transcriptase (RT) of PERV-B. The purified PERV RT was studied biochemically in comparison with the RT of murine leukemia virus (MLV), because of the high-sequence homology between these two RTs. The data show that in several properties the two enzymes are similar, particularly regarding the monomeric subunit composition of the proteins in solution, the high resistance to deoxynucleoside analogues, and the pattern of RNA cleavage by the ribonuclease H activity (RNase H) of the RTs. However, in several cases there are apparent differences between the two RTs, most notable the divalent cation preference (Mn+2 versus Mg+2) in the DNA polymerase reactions. As already shown for viral PERV RT, the novel recombinant PERV RT exhibits a relatively high resistance to several deoxynucleoside analogue inhibitors, suggesting that they might not be very efficient in inhibiting the replication of PERV virions. Therefore, the availability of large amounts of the recombinant RT can be useful for a wide screening of novel drugs against infectious PERV.

The human endogenous retroviruses (HERVs), ERV3 (HERV-R) and HERV-K, are both known to be transcriptionally active in human placenta. In the case of ERV3 there is also indirect evidence for its participation in cellular differentiation. In this study we examined the expression of ERV3 (HERV-R) and HERV-K in human normal fetal tissues by in situ hybridization. The highest level of ERV3 env expression was detected in primitive adrenal cortex. Elevated levels of expression were also found in the following developing tissues: kidneys (tubules), tongue, heart, liver, and central nervous system. Tissue-specific expression was found for HERV-K rec (former cORF) but not for pol/int transcripts. The highest rec expression was found in placenta and levels slightly higher than sense control were found in the rest of the tissues examined. Pol/Int was not possible to quantitate. It appears that ERV3 is expressed in an organ-specific way during embryogenesis and might suggest a possible role in the development and differentiation of human tissues.

Vertically transmitted endogenous retroviruses pose an infectious risk in the course of pig-to-human transplantation of cells, tissues, and organs. Two classes of polytropic type C porcine endogenous retroviruses (PERV) productively infect human cells in vitro. The cloning and characterization of replication-competent PERV-B sequences from infected human cells (F. Czauderna, N. Fischer, K. Boller, R. Kurth, and R. R. Tönjes, J. Virol. 74:4028-4038, 2000) as well as the cloning of functional PERV-A and -B sequences from porcine cell line PK15 (U. Krach, N. Fischer, F. Czauderna, and R. R. Tönjes, J. Virol. 75:5465-5472, 2001) have been previously described. Here we report the isolation of four full-length proviral sequences from a porcine bacterial artificial chromosome (BAC) library that comprises chromosomally assigned PERV. Clones Bac-PERV-A(130A12) and Bac-PERV-A(151B10) map to pig chromosome 1 and demonstrate close homology to PK15-PERV-A(58) in env and to PERV-MSL in long terminal repeat (LTR), gag , and pro/pol sequences. Clone Bac-PERV-A(463H12) is located on pig chromosome 3 and demonstrates close homology to PK15-PERV-A(58) in env and to 293-PERV-B(43) in LTR, gag , and pro/pol (Czauderna et al.; R. R. Tönjes, F. Czauderna, N. Fischer, U. Krach, K. Boller, P. Chardon, C. Rogel-Gailard, M. Niebert, G. Scheef, A. Werner, and R. Kurth, Transplant Proc. 32:1158-1161, 2000). Clone Bac-PERV-B(192B9) is located on pig chromosome 7 in the swine leukocyte antigen region and is highly homologous with but distinct from the previously described functional clone 293-PERV-B(43) and bears the number of repeats initially observed in the LTRs of clone 293-PERV-A(42) (Czauderna et al.; Krach et al.). Clones Bac-PERV-A(130A12), Bac-PERV-A(151B10), and Bac-PERV-A(463H12) were replication competent upon transfection into susceptible 293 and HeLa cells. Bac-PERV-B(192B9), however, bears two stop codons in pro/pol preventing this clone from being replication competent in some individual pigs, but initial screenings indicate that this provirus might be intact in others. The data suggest that the porcine genome harbors a limited number of infectious PERV sequences, allowing for specific screening in different pig breeds.

HLA-DQ genes, located in the human leukocyte antigen region on chromosome 6 p, are the main inherited factors predisposing to Type I (insulin-dependent) diabetes mellitus. Endogenous retroviral long-terminal repeats are integrated at several sites within this region, one of which is known to enhance susceptibility for Type I diabetes. We examined another LTR within the HLA-region as an additional genetic risk marker.We investigated the segregation of one long-terminal repeat (DQ-LTR13), located 1.3 kb upstream of HLA DQB1 with different HLA-DQ haplotypes, and its transmission to patients. A total of 284 Caucasian families (203 German and 81 Belgian) with at least one diabetic offspring were genotyped for DQA1, DQB1 and DQ-LTR13.DQ8/LTR13(+) was preferentially transmitted (139 transmitted vs 28 not transmitted; P(TDT) = 1.67 x 10(-14)) whereas no deviation from expected transmission frequencies was observed for DQ8/LTR13(-) (20 transmitted vs 17 not transmitted; P(TDT) = 1.00). DQ8/LTR13(+) alleles conferred a significantly higher risk for Type I diabetes than DQ8/LTR13(-) alleles (p chi(2) = 2.58 x 10(-14)). This difference remained significant even after DRB1 subtyping (p chi(2) = 0.02). Also, there was a significant difference when comparing the transmission of DQ2/LTR13(+) and DQ2/LTR13(-) alleles (p chi(2) = 0.01), the latter conferring an increased risk. The transmission of DQ-LTR13(+) haplotypes did not show any differences regarding paternal, maternal or gender-related stratification. However, DQ8/LTR13(-) was significantly more often transmitted from mothers (p chi(2) = 0.01) and to female patients (p chi(2) = 0.04).We conclude that DQ-LTR13 marks additional genetic risk for Type I diabetes on predisposing DRB1(*)0401- DQ8 and DQ2 haplotypes and will help to further define susceptibility in this gene region.

Solid organ and cell transplantation, including pancreatic islets constitute the treatment of choice for chronic terminal diseases. However, the clinical use of allogeneic transplantation is limited by the growing shortage of human organs. This has prompted us to initiate a unique multi-center and multi-team effort to promote translational research in xenotransplantation to bring xenotransplantation to the clinical setting. Supported by the German Research Foundation, an interdisciplinary group of surgeons, internal medicine doctors, diabetologists, material sciences experts, immunologists, cell biologists, virologists, veterinarians, and geneticists have established a collaborative research center (CRC) focusing on the biology of xenogeneic cell, tissue, and organ transplantation. A major strength of this consortium is the inclusion of members of the regulatory bodies, including the Paul-Ehrlich Institute (PEI), infection specialists from the Robert Koch Institute and PEI, veterinarians from the German Primate Center, and representatives of influential ethical and religious institutions. A major goal of this consortium is to promote islet xenotransplantation, based on the extensive expertise and experience of the existing clinical islet transplantation program. Besides comprehensive approaches to understand and prevent inflammation-mediated islet xenotransplant dysfunction [immediate blood-mediated inflammatory reaction (IBMIR)], we also take advantage of the availability of and experience with islet macroencapsulation, with the goal to improve graft survival and function. This consortium harbors a unique group of scientists with complementary expertise under a cohesive program aiming at developing new therapeutic approaches for islet replacement and solid organ xenotransplantation.

Vertically transmitted endogenous retroviruses pose an infectious risk in the course of pig-to-human transplantation of cells, tissues, and organs. Two classes of polytropic type C porcine endogenous retroviruses (PERV) which are infectious for human cells in vitro are known. Recently, we described the cloning and characterization of replication-competent PERV-B sequences from productively infected human cells (F. Czauderna, N. Fischer, K. Boller, R. Kurth, and R. R. Tönjes, J. Virol. 74:4028–4038, 2000). Here, we report the isolation of infectious molecular PERV-A and PERV-B clones from pig cells and compare these proviruses with clones derived from infected human 293 cells. In addition to clone PERV-A(42) derived from 293 cells, four “native” full-length proviral PERV sequences derived from a genomic library of the porcine cell line PK15 were isolated. Three identical class A clones, designated PK15-PERV-A(42), PK15-PERV-A(45), and PK15-PERV-A(58), and one class B clone, PK15-PERV-B(213), were characterized. PK15-PERV-B(213) is highly homologous but distinct from the previously described clone PERV-B(43). PK15-PERV-A(58) demonstrates close homology to PERV-A(42) in env and to PERV-C in long terminal repeat, gag , and pro / pol sequences. All three PERV clones described here were replication competent upon infection of susceptible cell lines. The findings suggest that the pig genome harbors a limited number of infectious PERV-A and -B sequences.

The polymerase enhanced reverse transcriptase (PERT) assay is a highly sensitive assay for the detection of reverse transcriptase (RT) activity in culture supernatants of retrovirus-producing cells. However, some cellular DNA-dependent DNA polymerases exhibit RT-like activities in this assay. A synthetic DNA competitor which suppresses the RT-like activities of cellular DNA-dependent DNA polymerases was used in a modified PERT assay technique for specific detection of RT activity in culture supernatants of retrovirus-producing cells. We determined the optimum condition of the assay and evaluated its specificity. This improved PERT assay is easy to perform and is able to detect minute amounts of purified RT, as well as RT in crude cell lysates and concentrated culture supernatants.

Long terminal repeats (LTRs) are common retrovirus-related sequences spread throughout the human genome. We previously reported the human-specific integration of one LTR (DQLTR3) located 15 kb upstream of HLA DQB1. To elucidate the contribution of retroviral sequences to the variability and phylogenetic background of HLA DQB1 we investigated another LTR (DQLTR13), located 1.3 kb upstream of HLA DQB1, in German families, great apes, and Old World monkeys. Within German families, DQLTR13 presence was strongly linked to HLA DQB1*0302, *0303, and *0402 haplotypes. All other haplotypes had a low frequency or were devoid of DQLTR13. Phylogenetic analysis of DQLTR13 and adjacent nucleotide sequences in humans and non-human primates revealed a high degree of similarity and recent origin of HLA DQB1*0302, *0303, and *0402. Nevertheless, two lineages leading to DQB1*0301 and *0302 were generated by an ancient split of a DQB1*0301, *0302 progenitor. A third lineage consisting of DQB1*05/*06-related sequences may have evolved from the DQB1*0302 lineage, and a DQB1*0201-related sequence shared common ancestry with DQB1*0301. Among the human haplotypes, HLA DQB1*0201 and *0301 are linked to two different DQA1 alleles. Based on the small genetic distance of DQLTR13 as well as the adjacent sequences on these haplotypes, we suggest that a recent recombination is responsible for these associations. In the analysis of nonhuman primate species, we detected DQLTR13 in two lowland gorillas, dating the integration at at least 8 million years ago. We therefore conclude that noncoding sequences up to 1.3 kb upstream of DQB1 provide novel insight into the generation of MHC gene diversity.

Advances in xenotransplantation offer chances to alleviate the shortage of human donor organs. The discovery that pig endogenous retroviruses (PERV) can infect human cells in vitro has stimulated the discussion on infectious risk in xenotransplantation. A molecular and immunologic monitoring of xenograft recipients and of donor animals for putative infection with PERV and other microorganisms is inevitable. In this report, we describe the generation and testing of a highly specific anti-serum directed against the PERV nucleocapsid protein. The Gag amino acid (aa) sequence of PERV class B was used to define immunogenic domains by computer analysis. A peptide corresponding to the C-terminal 19 aa of the 10 kDa (p10) nucleocapsid (NC) portion of the Gag polyprotein was used to immunize rabbits. The generated serum was tested using recombinant PERV Gag protein expressed in insect cells, purified PERV virus particles and human 293 cells transfected or infected with PERV, respectively. Test methods included Western blotting, indirect immunofluorescence, immunoperoxidase assay and ELISA. The PERV anti-serum provides a tool that is instrumental for detection of a potential agent of zoonosis. It can be used for screening of donor animals and xenograft recipients in the course of xenotransplantation procedures.

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The human genome harbors 25 to 50 proviral copies of the endogenous retrovirus type K (HERV-K), some of which code for the characteristic retroviral proteins Gag, Pol, and Env. For a genome-wide cloning approach of full-length and intact HERV-K proviruses, a human P1 gene library was screened with a gag -specific probe. Both HERV-K type 1 and 2 clones were isolated. Sixteen HERV-K type 2 proviral genomes were characterized by direct coupled in vitro transcription-in vitro translation assays to analyze the coding potential of isolated gag , pol , and env amplicons from individual P1 clones. After determination of long terminal repeat (LTR) sequences and adjacent chromosomal integration sites by inverse PCR techniques, two HERV-K type 2 proviruses displaying long retroviral open reading frames (ORFs) were assigned to chromosomes 7 (C7) and 19 (C19) by using a human-rodent monochromosomal cell hybrid mapping panel. HERV-K(C7) shows an altered (YIDD-to-CIDD) motif in the reverse transcriptase domain. HERV-K(C19) is truncated in the 5′ LTR and harbors a defective protease gene due to a point mutation. Direct amplification of proviral structures from single chromosomes by using chromosomal flanking primers was performed by long PCR for HERV-K(C7) and HERV-K(C19) and for type 1 proviruses HERV-K10 and HERV-K18 from chromosomes 5 and 1, respectively. HERV-K18, in contrast to HERV-K10, bears no intact gag ORF and shows close homology to HERV-K/IDDMK 1,2 22. In transfection experiments, HERV-K(C7) and HERV-K cDNA-based expression vectors yielded the proteins Gag and cORF whereas HERV-K10 vectors yielded Gag alone. The data suggest that the human genome does not contain an entire, intact proviral copy of HERV-K.

Long terminal repeats (LTRs) of the human endogenous retrovirus K (HERV-K) family have been found at several sites within the human genome, of which one is located in the vicinity of HLA-DQB1. Since this DQLTR3 is only present on some haplotypes, we performed a linkage analysis in 130 Caucasian families. In order to date the integration event we also investigated the presence of this DQLTR3 in apes and Old World monkeys. Additionally, we sequenced the adjacent region of DQLTR3-positive and -negative haplotypes in humans, apes and old world monkeys to elucidate their evolution. Linkage analysis revealed a differential integration of DQLTR3 on specific HLA-DQ haploypes: there was a high frequency of this LTR on haplotypes containing HLA-DQB1*0302 (0.96) and a moderate frequency on HLA-DQB1*0402 (0.78), HLA-DQB1*0303 (0.44), HLA-DQB1*0502 (0.38) and HLA-DQB1*0301 (0.35). HLA-DQB1*0201 (0.18), HLA-DQB1*0503 (0.15), HLA-DQB1*0603 (0.15), HLA-DQB1*0602 (0.04), HLA-DQB1*0501 (0.03) and HLA-DQB1*0604 were rarely positive or devoid of DQLTR3. In apes and Old World primates there was no DQLTR3 rendering it a human specific insertion. Sequence analysis of the adjacent region showed two different motifs in humans corresponding to either presence or absence of DQLTR3. Two different motifs were observed within three sequences of Macaca mulatta: One motif is closely related to the sequence from Macaca nemestrina and Macaca fascicularis whereas the other sequence is more closely related with that of Papio papio and Cercopithecus aethiops. Therefore the analysis of retroviral elements as well as intronic sequences of MHC-DQB1 could help to clarify the evolution of this gene region as well the phylogenic relationship between humans, apes and Old World monkeys.

Major genetic susceptibility to type 1 diabetes mellitus maps to the human leukocyte antigen (HLA) region on chromosome 6p. During evolution, endogenous retroviral long terminal repeats (LTR) have been integrated at several sites within this region. We analyzed the presence of a solitary HERV-K LTR in the HLA DQ region (DQ-LTR3) and its linkage to DRB1, DQA1, and DQB1 haplotypes derived from 246 German and Belgian families with a patient suffering from type 1 diabetes mellitus. Segregation analysis of 984 HLA DQA1/B1 haplotypes showed that DQ-LTR3 is linked to distinct DQA1 and DQB1 haplotypes but is absent in others. The presence of DQ-LTR3 on HLA DQB1*0302 haplotypes was preferentially transmitted to patients from heterozygous parents (82%; P < 10(-6)), in contrast to only 2 of 7 DQB1*0302 haplotypes without DQ-LTR3. Also, the extended HLA DRB1*0401, DQB1*0302 DQ-LTR3-positive haplotypes were preferentially transmitted (84%; P < 10(-6)) compared with 1 of 6 DR-DQ matched DQ-LTR3 negative haplotypes. DQ-LTR3 is missing on most DQB1*0201 haplotypes, and those LTR3 negative haplotypes were also preferentially transmitted to patients (80%; P < 10(-6)), whereas DQB1*0201 DQ-LTR3-positive haplotypes were less often transmitted to patients (36%). Other DQA1/B1 haplotypes did not differ for DQ-LTR3 between transmitted and nontransmitted haplotypes. Thus, the presence of DQLTR3 on HLA DQB1*0302 and its absence on DQB1*0201 haplotypes are independent genetic risk markers for type 1 diabetes.

The human hemopoietic cell kinase (HCK) is a member of the src family of protein tyrosine kinases specifically expressed in myeloid cells and to a minor extent in B-lymphoid cells. HCK expression is up-regulated at the transcriptional level during myeloid differentiation of hematopoietic cells. To elucidate the molecular basis of the differential HCK gene expression, the genomic region containing the HCK promoter was isolated and functionally characterized. A DNA fragment containing 101 base pairs of the 5'-flanking sequence showed strong promoter activity in the macrophage cell line RAW264 but was inactive in the non-monocytic cell lines HUT-78 and NIH-3T3. Site-directed mutagenesis of the proximal promoter region showed that two GC-rich sequence elements are essential for transcriptional activity in myeloid cells. Electrophoretic mobility shift analysis using nuclear extracts obtained from RAW264 cells and from the promonocytic cell line U-937 revealed the formation of at least three distinct protein-DNA complexes at each of these sites, one of which was found to contain the transcription factor Sp1. Expression of a reporter gene linked to the -101 HCK promoter region was up-regulated by Sp1, but not by other members of the Sp1 family of transcription factors, in Drosophila Schneider cells. A synergistic effect on HCK promoter activity was observed at high concentrations of Sp1. Our results show that Sp1 plays an essential role in the regulation of the differential gene expression of the HCK gene.

The human endogenous retrovirus type K (HERV-K) family codes for the human teratocarcinoma-derived retrovirus (HTDV) particles. The existence of the envelope protein (ENV) of HERV-K encoded by the subgenomic env mRNA has not yet been demonstrated. To study the genetic requirements for successful expression of ENV, we have constructed a series of recombinant HERV-K env expression vectors for infection and transfection experiments in insect cells and mammalian cells, respectively. Six baculovirus constructs bearing full-length or truncated HERV-K env with or without homologous or heterologous signal peptides were used for infections of insect cells. All recombinant baculoviruses yielded ENV proteins with the expected molecular masses. The full-length 80- to 90-kDa HERV-K ENV protein including the cORF leader sequence was glycosylated in insect cells. In addition, the 14-kDa cORF protein was expressed due to splicing of the full-length env mRNA. The ENV precursor protein is not cleaved to the surface (SU) and transmembrane (TM) glycoproteins; it does not appear on the surface of infected insect cells and is not secreted into the medium. For ENV expression in COS cells, plasmid vectors harboring the cytomegalovirus immediate-early promoter/intron A element and the tissue plasminogen activator (t-PA) signal peptide or the homologous HERV-K signal peptide upstream of the env gene were employed. Glycosylated and uncleaved ENV was expressed as in GH teratocarcinoma cells but at higher levels. The heterologous t-PA signal sequence was instrumental for expression of HERV-K ENV on the cell surface. Hence, we have shown for the first time that the HERV-K env gene has the potential to be expressed as a full-length envelope protein with appropriate glycosylation. In addition, our data provide explanations for the lack of infectivity of HERV-K/HTDV particles.

The linker histone H1degree accumulates in terminally differentiating cells and replaces other members of the H1 histone family, even in the absence of cell division. To study the role of H1degree in vivo, we have created two lines of transgenic mice with either the human H1degree promoter (HH minigene) or the mouse metallothionein T promoter (MH minigene) upstream of the human H1degree gene. Mice bearing the minigenes HH or MH overexpress human H1degree mRNA at 10-20-fold higher levels than in normal mice in a constitutive or metal-inducible manner. In contrast to this increase in mRNA content, which was studied in liver, kidney and brain, no significant changes in the relative proportions of the H1 protein subtypes, including H1degree were observed. Transgenic mice exhibited normal anatomic phenotypes, growth rates and reproduction rates. Thus, our results suggest a posttranscriptional and/or translational mechanism that compensates the unbalanced linker-histone expression in different tissues.

All primates studied to date produce retroviral-like particles in their placentae. We have purified these particles from two primate species, one Old World (human) and one New World (marmoset), and have identified the retroviral sequences which are packaged into these particles. Three families of sequences have been detected in these particles in human, all of which have the highest homology to B- and D-type retroviruses and to the human endogenous retrovirus HERV-K10. Previous studies have reported that the New World monkeys do not possess sequences with homology to HERV-K10. We have identified a new family of low-copy-number sequences which are present in New World monkeys and which possess 70% homology to the HERV-K family. Particles from both species possess reverse transcriptase activity and we have found that some of these retroviral particles package sequences which encode long open reading frames inpol,as revealed by expression cloning inEscherichia coli.These open reading frames could encode the reverse transcriptase enzyme activity found in the particles.