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Work Package 1 – Endothelial Cell Activation
A number of novel genes were cloned and have been evaluated in our in-vitro systems to the extent that important steps have already been decided. As far as TTP, a more comprehensive analysis has revealed a previously uncharacterized function in NF-kB signalling. A recombinant adenoviral vector for TTP has been generated that allows tet-regulated expression. LNK has been characterised for activation and survival pathways, and a search for molecular ligands has generated a link to the integrin pathway. Furthermore, LNK was characterised with regard to protection against xenogeneic natural antibodies-mediated vascular responses. Similar studies on ADAM10 are under way. Identification of Nrf2 target genes and their characterisation was delayed due to instability of the protein. However, a strategy to overcome this problem has been identified and this is expected to result in the identification of novel protective genes. Concerning coagulation studies, the contribution and interplay between EPCR and TM (human vs. porcine) was further studied. Our results further strengthen the importance of human TM. Studies on non-gal xenoreactive antibodies and their elicited signalling pathways have continued.
Collectively, the very promising efficacy data generated in vitro using porcine endothelial cells (EC) support the view that expression of some of these genes currently under investigation, especially TTP and LNK, in transgenic pig organs will confer protection to porcine xenografts transplanted into primates. In this light, the decision was taken by the XENOME Project Managment Board to transfer such constructs to WP3 (Pig-to-Primate) to prepare the ground for the production of transgenic pigs on a GalKO-/-CD55+/+ background expressing both TTP and LNK.
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Work Package 2 – Safety
The PERV epigenetics study, in conjunction with the integration study, has identified a PERV-C locus which is hypomethylated and is likely responsible for human-tropic infectious (HTRC) PERV in miniswine. These results strongly support our hypothesis that PERV methylation is a key factor influencing PERV expression leading to production of HTRC PERV. We are now extending this study to XENOME GalT-KO pigs to assess the presence of active proviruses. PERV suppressor constructs containing anti-Gag shRNA have been developed jointly with WP3 and Joachim Denner (RKI, Berlin) and are now ready for animal engineering. As an alternative strategy, a restriction factor, tetherin, which targets PERV release, showed significant effects in cell culture assays. Because this task has a high priority in XENOME safety aspects, search for PERV suppressors will continue and expand. We have also adjusted our focus in the PERV integration study to verify solo-LTR/intact status and prevalence of sites already obtained. The solo-LTR/intact status has been completed for PERV-C and the work on PERV-A/B is well underway. The presence/prevalence of full length PERV at these insertion sites will be verified in various pig lines, including XENOME GalT-KO transgenic pigs. Screening for PERV and non-PERV agents has continued in collaboration with WP3 and WP4. PCR testing for HEV, PCMV, PCV-2, and PLHV-1 has shown the presence of these pathogens in most of the herds tested. No evidence of PERV infection was detected in the xenografted patients receiving pigskin analysed to date. The recruitment of additional patients is underway.
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Work Package 3 – Genetic Engineering of Pigs
Considerable progress has been made by WP3 partners since the last reporting period. Large number of homozygous Gal KO (Gal-/-) pigs were produced via somatic cloning and successful attempts were made to add further transgenes on this genetic background. LTR-AVT produced a total of 105 Gal-/- piglets, 57 of which alive (3 from NSRRC/ CD55-CD39 line [also known as NSRRC/XENOME line] and 54 derived from the Australian (Gal-/-/CD55/CD59/CD39/HTF) line. Moreover, two stillborn DAF-CD39 piglets from the Sachs line modified in the LTR-AVT lab were produced. A new hTM expression vector was co-transfected with EPCR into Gal-/- porcine aortic endothelial cells (PAEC) and supplied to UPD for analysis. Western Blotting and Real-Time PCR were employed to increase the accuracy in the selection of fibroblasts prior to nuclear transfer. FLI produced Gal-/- piglets from the NSRRC source and continued the breeding program with the available Gal-/+ (Max) line.
However, no live Gal-/- piglets could be obtained from the latter line and it has been decided to stop the work on the Max line. Indeed, from now onwards, the NSRRC/XENOME line, that expresses CD55 on a Gal-KO background, will replace the Max line and will be used for all the genetic engineering for the remainder of the project. In this regard, the new pig breeding strategy for the remaining two years of XENOME and the fundamental approaches that will be followed are described in details in Tables 1 and 2 in the Addendum to the description of WP3. It should be noted that results unequivocally demonstrate that the PAEC deriving from the NSRRC/XENOME line express an amount of CD55 greater than that observed on the human counterpart (HUVEC and HAEC). In the current years the study on the production and characterisation of hA20 pigs has been completed. A new TFPI construct was made, validated and used in somatic cell nuclear transfer. The characterisation of the recently-born piglets is under way. A promising TF siRNA vector was developed and it will be used in the next period. UNIMIB expanded the work on integrative and episomal vectors for sperm-mediated gene transfer (SMGT). Several multiple transgenic piglets were produced; however, transgenic expression levels were unsatisfactory. Uptake of foreign DNA into semen from Gal-/+ boars was studied in detail and revealed similar kinetics to wild-type semen. MHH developed and validated in-vitro assays for assessing the biological function of hTM, hA20 and HOXA9. These assays demonstrate that hTM and hA20 are functionally expressed in transgenic pigs. Members of WP3 reported results of their research at international meetings and in peer-reviewed publications.
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Work Package 4 – Pig-to-Primate
The ready availability of source pigs in the third year has enabled significant progress in the animal models developed by WP4. Indeed, considerable advances have been made with regard to the characterisation of the elicited anti non-Gal antibody repertoire in xenografted primates. By using bidimensional Western blotting, protein spots excision and mass spectrometry, several targets recognised by the elicited immune response could be identified (Caldesmon (CALD1), ATP synthase subunit beta, Enolase, Tubuline alpha chain, vimentin, Phosphopyruvate hydratase (fragment) and Proliferating cell nuclear antigen). Further analyses are underway to identify whether these could represent hypothetical targets of intervention for the colleagues in WP3. With regard to renal xenotransplantation, xenografts from GalT-KO/CD55/CD59/CD39/HT transgenic pigs were transplanted into immunosuppressed baboons and cynomolgus monkeys. Survival was not improved in these animals with acute humoral rejection (AHXR) being the first cause of graft loss in the presence of high titers of elicited anti non-gal anti-pig antibodies. As a large number of B cells was unexpectedly observed in rejected grafts, cyclophosphamide in the induction regimen was replaced with rituximab. Interestingly, the use of this agent was able to entirely negate the elicited anti non-Gal anti-pig antibody response, but could not extend xenograft survival. As a consequence, new studies are already planned to explore novel immunosuppressive strategies (such as bortezomib) to improve survival in our models. As far as islet xenotransplantation is concerned, the data generated in year 3 unequivocally demonstrate that cynomolgus monkeys that have previously lost pig islets inserted in a subcutaneous monolayer cellular device (MCD) can safely undergo retransplantation with a new set of islet with excellent clinical results (reversal of diabetes with HbA1c comprised between 7.4 and 9.6%). Finally, our neuron xenotransplantation studies using neural precursors from CTLA4-Ig pigs have reproducibly and convincingly demonstrated that transplantation of this type of cellular xenograft is associated with long term survival when transplanted into Parkinsonian immunosuppressed cynomolgus monkeys. More importantly, neural xenograft survival is associated with evident clinical recovery (significant improvement of Parkinson’s disease), evident survival and function of the porcine graft as demonstrated by PET imaging and evident graft survival at autopsy (more than 6 months after transplantation). The outcome of these experiments is spectacular and represented one of the highlights of the IXA 2009 Venice conference. In all cases, PERV infections were not observed in our 3 pig-to-primates xenotransplantation models.
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Work Package 5 – Ethical and Legal Aspects
Several reports have been completed. These address different critical issues related to xenotransplantation and include the moral and legal status of animals; a comparison amongst different regulatory models; and a critical comparative analysis of the regulatory aspects with regard to safety issues. The reports have now been circulated amongst partners for their comments and revision. Once reviewed, they will be posted on the XENOME website. WP5 has also finished a discussion paper aimed at determining where community support and concerns lie in relation to the complex issues surrounding xenotransplantation. The aim of this paper is to facilitate a public discussion on xenotransplantation research, including the relevant ethical and safety issues. Once reviewed by the partners, it will also be posted on the web as a useful tool to prepare recommendations for the regulation of xenotransplantation in Europe. Public comments will be welcome. The public consultation has been held by a subcontractor, Observa, with WP6 in five representative EU countries (namely Lithuania, Italy, the UK, Bulgaria, and the Czech Republic). The results of these activities will be of help in the development of draft guidelines and recommendations with regard to xenotransplantation.
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Work Package 6 – Dissemination, Demonstration and Training
All the sections of the XENOME web site have been completed and most of them constantly updated throughout the year. In particular, the Dissemination Area now provides materials on different events, publications and press releases. The Legal and Ethical Area was also updated with new bibliographic references and with the inclusion of a detailed analysis of the regulatory frameworks existing in the various EU and non-EU countries. Several teaching events related to the project have been organised, in particular, at Venice. Finally, Observa has already conducted the consultation in five representative countries across the European Union. The analysis of the acquired data is currently underway
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