Abstract: 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 WHA 57.18 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.

Abstract: 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.

Abstract: Xenotransplantation may be a solution to overcome the shortage of organs for the treatment of patients with organ failure, but it may be associated with the transmission of porcine microorganisms and the development of xenozoonoses. Whereas most microorganisms may be eliminated by pathogen-free breeding of the donor animals, porcine endogenous retroviruses (PERVs) cannot be eliminated, since these are integrated into the genomes of all pigs. Human-tropic PERV-A and -B are present in all pigs and are able to infect human cells. Infection of ecotropic PERV-C is limited to pig cells. PERVs may adapt to host cells by varying the number of LTR-binding transcription factor binding sites. Like all retroviruses, they may induce tumors and/or immunodeficiencies. To date, all experimental, preclinical, and clinical xenotransplantations using pig cells, tissues, and organs have not shown transmission of PERV. Highly sensitive and specific methods have been developed to analyze the PERV status of donor pigs and to monitor recipients for PERV infection. Strategies have been developed to prevent PERV transmission, including selection of PERV-C-negative, low-producer pigs, generation of an effective vaccine, selection of effective antiretrovirals, and generation of animals transgenic for a PERV-specific short hairpin RNA inhibiting PERV expression by RNA interference.

Abstract: Pig to human xenotransplantation represents an ambitious venture that requires, besides evasion of rejection mechanisms and physiological incompatibilities, the generation of pathogen‐free pigs as donors for well characterized xenografts to warrant medicinal products that do comply with statutory regulatory demands [1–3]. The publication of a high quality draft sequence for the pig genome (Sus scrofa) and a series of accompanying reports for the first time offered the feasibility of whole genome expression profiling of porcine tissues and cells [4–8] . The SFB TR CRC 127 project Z2 “Microbiological Safety including Virological Safety” is based on microbial profile analysis of porcine tissues in order to prevent zoonotic events, including infection by porcine endogenous retroviruses (PERV) [9]. The project comprises the detection and characterization of potential pathogens as well as the investigation of the microbial influence on the transcriptional status of tissues and cells. Hence, specific expression patterns, e.g. up‐regulation of antiviral host factors or cell cycle/apoptotic regulators, may also provide information on ongoing or precedent events that may have impact on tissues/cells quality and therefore its suitability as xenografts. We use microarray technology for monitoring viability of tissues and cells as well as their microbial/viral status. An Agilent based, 60K DNA microarray representing 25,415 different genes of the recently published Sus scrofa genome (NCBI Sus scrofa 10.2‐assembly) was generated [10]. The microarray was specified for German Landrace and Göttingen Minipig, amongst other pig species, by hybridizing complex RNA samples generated from five different pig organs and blood as well as chromosomal porcine DNA to highlight non expressed genes. Four Diagnostic PERV sequences for pro/pol (all classes of PERV), env (to differentiate between PERV‐A, ‐B and ‐C) as well as 15 human transgenes such as CD59 (human complement regulatory protein), DAF (Decay accelerating factor or CD55), human A20 (hA20) and others were included. In total, the microarray displays 25,434 genes each represented by up to three different 60‐mer oligonucleotides. To reveal functionality of the microarray the transcriptional status of ST‐IOWA cells freshly infected with molecularly cloned virus PERV‐C (1312) [11] was monitored. Total mRNA levels at day 7, 28 and 56 post infection were compared with naive uninfected cells. All samples were tested in triplicates and the relative signal intensity of hybridized probes was compared. Special attention was given to antiviral host factors such as APOBEC and tetherin of which involvement as antiviral factors on PERV expression has been demonstrated [12–14] . Constitutively expressed housekeeping genes, i.e. porcine glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), beta actin and cyclophilin A, respectively, were used as controls [15, 16]. The presented microarray supports the safety and quality by monitoring the transcriptional status of xenotransplants. References [1] EMEA/CHMP/CPWP/83508/2009. Guideline on Xenogeneic Cell‐Based Medicinal Products. [2] U.S. Department of Health and Human Services. Food and Drug Administration. Center for Bi ologics Evaluation and Research (CBER). February 2002. Draft Guidance for Industry: Precautionary Measures to Reduce the Possible Risk of Transmission of Zoonoses by Blood and Blood Products from Xenotransplantation Product Recipients and Their Intimate Contacts. [3] U.S. Department of Health and Human Services. Food and Drug Administration. Center for Biologics Evaluation and Research (CBER). April 2003. Guidance for Industry: Source Animal, Product, Preclinical, and Clinical Issues Concerning the Use of Xenotransplantation Products in Humans. [4] Groenen MA, et al. Analyses of pig genomes provide insight into porcine demography and evolution. Nature 2012; 491(7424): 393–398. [5] Li Y, Mei S, Zhang X, et al. Identification of genome‐wide copy number variations among diverse pig breeds by array CGH.BMC Genomics 2012; 13:725. doi: 10.1186/1471‐2164‐13‐725 . [6] Servin B, Faraut T, Iannuccelli N, Zelenika D, Milan D. High‐resolution autosomal radiation hybrid maps of the pig genome and their contribution to the genome sequence assembly. BMC Genomics 2012; 13:585. doi: 10.1186/1471‐2164‐13‐585 . [7] Nguyen DT, Lee K, Choi H, et al. The complete swine olfactory subgenome: expansion of the olfactory gene repertoire in the pig genome. BMC Genomics 2012; 13:584. doi: 10.1186/1471‐2164‐13‐584 . [8] Uenishi H, Morozumi T, Toki D et al. Large‐scale sequencing based on full‐length‐enriched cDNA libraries in pigs: contribution to annotation of the pig genome draft sequence. BMC Genomics 2012; 13:581. doi: 10.1186/1471‐2164‐13‐581 . [9] Denner J, Tönjes RR. Infection barriers to successful xenotransplantation focusing on porcine endogenous retroviruses. Clin Microbiol Rev. 2012; 25(2): 318–743. [10] Agilent's Microarray Platform. How High‐Fidelity DNA Synthesis Maximizes the Dynamic Range of Gene Expression Measurements. Library – Application Note. 2013; Publication Part Number: 5989‐9159EN [11] Preuss T, Fischer N, Boller K, Tönjes RR. Isolation and characterization of an infectious replication‐competent molecular clone of ecotropic porcine endogenous retrovirus class C. J Virol. 2006; 80(20):10258–61. [12] Dörrschuck E, Fischer N, Bravo IG, et al. Restriction of porcine endogenous retrovirus by porcine APOBEC3 cytidine deaminases. J Virol. 2011; 85(8):3842–57. [13] Dörrschuck E, Münk C, Tönjes RR. APOBEC3 proteins and porcine endogenous retroviruses. Transplant Proc. 2008; 40(4):959–61. [14] Mattiuzzo G, Ivol S, Takeuchi Y. Regulation of porcine endogenous retrovirus release by porcine and human tetherins. J. Virol. 2010; 84: 2618 –2622. [15] Mcculloch RS, S. Ashwell M, O'Nan AT, Mente PL. Identification of stable normalization genes for quantitative real‐time PCR in porcine articular cartilage. J Anim Sci Biotechnol. 2012; 3(1):36. [16] Nygard AB, Jørgensen CB, Cirera S, Fredholm M. Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR. BMC Mol Biol. 2007; 8:67.

Abstract: Porcine induced pluripotent stem cells (pi PSC s) offer an alternative strategy in xenotransplantation ( XT x). 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 ( pORF 1 and pORF 2) in different pi PSC ‐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 log 2 expression ratio 〉 1, and 4072 targets showed log 2 expression ratio less than −1 with 0.05 P ‐value threshold. Approximately ten percent of the targets showed highly significant expression ratios with log 2 ≥4 or ≤−4. Besides this general switch in cellular gene expression that was accompanied by an altered morphology, expression of both PERV and L1 pORF 1/ pORF 2 was significantly enhanced. pi PSC ‐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.

Abstract: 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.