Gene Therapy (2008) 15, 1247–1256 & 2008 Macmillan Publishers Limited All rights reserved 0969-7128/08 $32.00 www.nature.com/gt REVIEW Progress and prospects: genetic engineering in xenotransplantation S Le Bas-Bernardet1, I Anegon1 and G Blancho1,2 1INSERM, U643, Nantes, France; CHU Nantes, Institut de Transplantation et de Recherche en Transplantation, ITERT, Nantes, France; Universite´ de Nantes, Faculte´ de Me´decine, Nantes, France and 2Service de Ne´phrologie, Immunologie Clinique et Transplantation, CHU Nantes, Nantes, France In this review, we summarize the work published over the last the knockout of the a1,3-galactosyltransferase gene, fol- 2 years using genetic modifications of animals in the field of lowed by genetic engineering aimed at reducing the humoral xenotransplantation. Genetic engineering of the donor has and cellular immune response, complement activation and become a powerful tool in xenotransplantation, both for the coagulation. Finally, we report on the genetic modification of inactivation of one particular porcine gene and for the pigs to reduce porcine endogenous retrovirus infection risk addition of human genes with the goal of overcoming in the xenogeneic context. xenogeneic barriers. We summarize the work relative to Gene Therapy (2008) 15, 1247–1256; doi:10.1038/gt.2008.119 Keywords: aGal; knockout; xenoantibodies; transgenes; complement; xenotransplantation In brief Progress Prospects Genetic engineering approaches leading to a reduc- Deletion of the iGb3S gene in addition to deletion of tion of aGal expression in donor cells/organs and GalT in donor cells/organs. production of a1,3-galactosyltransferase knockout Crossbreeding of GalT-KO pigs with pigs transgenic (GalT-KO) pigs. for EndoGalC. Control of complement through the use of transgenic Crossbreeding of GalT-KO pigs with pigs transgenic pigs for multiple complement regulatory proteins. for different human anticoagulant and/or comple- Genetic engineering for molecules that control coa- ment regulatory proteins to decrease humoral and gulation activation. cellular immunological barriers. Genetic engineering for molecules that modify the Use of pig pancreatic islets genetically modified to cellular xenogeneic immune response express transgenes that control the cellular xeno- Control of PERV expression in pig cells/organs by geneic immune response and coagulation. RNA interference. Controlling PERV expression in pig cells/organs used in xenotransplantation to reduce the risk of host infection. virus transmission to humans. Now, pigs are considered for the future as animals of choice (porcine valves Introduction used for years in humans, may not be considered as xenotransplantation as the tissue is devitalized before Transplantation is currently facing a problem of organ implantation). In fact, pigs have been identified as shortage; waiting lists are increasing with some of these potential donors, primarily because of their physiology patients dying before any proposition of an organ transplant. One solution could be to increase the donor and anatomy being similar to humans. Moreover, there ‘reservoir’ by using animal organs: that is, xenotrans- are no major ethical considerations regarding their use in plantation. In the past, non-human primates (NHPs) medicine. Finally, their potential has been increased by were used as organ donors for humans (1960–1992), but the application of the most recent techniques of genetic are no longer considered as potential donors essentially manipulation: transgenesis, gene transfer through viral for ethical reasons and for a high risk of endogenous vector and cloning through nuclear transfer. However, some significant problems remain regarding their use: immunological barriers and the risk of viral trans- Correspondence: Dr G Blancho, Nephrology Unit, INSERM U643- mission. Manipulations of the pig genome targeting the ITERT, CHU Hoˆtel Dieu, 30 boulevard Jean Monnet, 44093 Nantes, Cedex 1, France. human or NHP immune responses, coagulation dis- E-mail: [email protected] orders and viral risk are research approaches that will be Received 13 May 2008; revised 30 June 2008; accepted 1 July 2008 addressed in this review (Table 1). Gene Therapy 1248 Table 1 Progress in xenotransplantation through genetic engineering approaches Technique Molecule Models Study results References Donor/target cells Recipient/responder cells Genetic engineering approaches leading to a reduction of aGal expression in donor cells/organs and production of a1,3-galactosyltransferase knockout (GalT-KO) pigs Gene transfer EndoGalC EndoGalC transgenic mice Mice transgenic for EndoGalC present Watanabe et al.1 reduced aGal expression, but an abnormal phenotype Cloning, GnT-I Transfected pig cells with Human serum Transfection with a pigGnT-I(320) mutation Matsunami et al.2 direct mutagenesis pigGnT-I mutations reduced the antigenicity to human sera Gene inactivation, GalT iGb3 vs GalT-transfected GalT-KO mice aGal synthesized by iGb3S is as Milland et al.3 gene transfer iGb3S 293 cells immunogenic as GalT Genetic engineering in xenotransplantation Gene inactivation GalT GalT-KO pig Complementarity breeding and SCNT in Nottle et al.4 GalT-KO pig production Gene inactivation GalT GalT-KO pig heart Baboon No improvement in coagulation problems Ezzelarab et al.5 in the GalT-KO context 6 Gene inactivation GalT PBMC from WT vs GalT-KO Healthy human vs Allosensitized compared to unsensitized Hara et al. S Le Bas-Bernardet pig patients awaiting renal patients at no greater risk of humoral transplantation rejection to pig xenografts Control of complement through the use of transgenic pigs for multiple complement regulatory proteins 7 DNA sequencing, hDAF hDAF transgenic pig heart Cynomolgus A restricted family IgVH3 gene encoded for Zahorsky-Reeves et al. gene transfer, their XNA et al phagemid vector Gene transfer hCD59 hCD59 transgenic Pig Microinjection of one hCD59 transgene into Deppenmeier et al.8 zygote pronuclei is a suitable technique to generate hCD59 transgenic pigs Gene transfer hCD55 Ex vivo perfused hCD55/59 Fresh human blood Xenotransplantation of lungs from hCD55 Poling et al.9 hCD59 transgenic porcine lung and hCD59 transgenic pigs induced, respectively, a partial protection and no effect on pulmonary dysfunction compared to nontransgenic lungs Gene transfer hDAF Pig heart Baboon Deregulated coagulation correlated closely Wu et al.10 hMCP with and probably causes primary failure of pig hearts transgenic for hCRP Gene transfer, hDAF Pig fibroblasts Human and baboon Much higher expression of hDAF might be Liu et al.11 plasmid sera beneficial in protecting pig organs from one humoral response SMGT hDAF Ex vivo perfused hDAF Fresh human blood Hearts from hDAF transgenic pigs Smolenski et al.12 transgenic porcine heart generated by SMGT are protected from HAR after contact with human blood Gene transfer, sCR1 Cultured human sCR1 Human AB serum Transgenic pECs expressing human sCR1 Manzi et al.13 plasmid transgenic porcine ECs are resistant to in vitro lysis by human serum Cloning, ICAM-2 Simian Virus 40-transformed The P8 sites associated with enhancer Godwin et al.14 gene transfer, promoter pig aortic endothelial cells activity contained in pig ICAM-2 pGL3-basic, (SVAP), COS-1 promoters seem to be crucial for achieving gene transfer, strong endothelial-specific transgene pRL-TK expression Table 1 Continued Technique Molecule Models Study results References Donor/target cells Recipient/responder cells Genetic engineering for molecules that control coagulation activation Gene transfer hCD39 In vitro islets isolated from Human blood Expression of transgenic hCD39 on Dwyer et al.15 hCD39 transgenic mice murine islets inhibits the in vitro clotting of human blood Genetic engineering for molecules that modify the cellular xenogeneic immune response Gene transfer, GalT (1) Autologous GalT-KO mice (1) Immunization of Transduction with lentiviral vectors results Mitsuhashi et al.16 lentiviral vector BM cells transfected with GalT-reconstituted in chimerism at sufficient levels to inhibit GalT mice (by BMT) with anti-aGal Ab production and to induce (2) Rabbit RBC rabbit RBC long-term tolerance under (2) GalT-BMT mice nonmyeloablative conditions transplanted with aGal mice hearts Gene transfer, GalT (1) Autologous Rhesus BM Immunization of Gene therapy achieves low-level, Fischer-Lougheed et al.17 lentiviral vector cells transfected with GalT aGalT+BM Rhesus long-term aGal chimerism sufficient to (2) Pig fibroblasts macaques with inhibit production of anti-aGal Ab after porcine cells immunization with porcine cells in rhesus macaques Gene transfer, CTLA4-Ig WT vs CTLA4-Ig-transduced Splenocytes from mice Production of CTLA4-Ig inhibits primed Mulley et al.18 lentiviral vector PIEC primed with PIEC indirect T-cell proliferation and cytokine responses in vitro S Le Bas-Bernardet Genetic engineering in xenotransplantation Cloning, PD-L1 Porcine PD-L1-transfected Human CD4+ T cells Porcine PD-L1 inhibited human activated Jeon et al.19 gene transfer CHO CD4+ T-cell proliferation inducing activated T-cell apoptosis Gene transfer HLA-E SCT PECs Human NK cells Transgenic expression of HLA-E SNCT Lilienfeld et al.20 induced a significant decrease of et al polyclonal human NK-mediated cytotoxicity against pEC but did not affect NK cell adhesion to pEC Control of PERV expression in pig cells/organs by RNA interference Gene transfer, PERV siRNA Pol2 transgenic Inhibition of PERV expression in Dieckhoff et al.21 lentiviral vector porcine cells