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The Applications of Genome Editing in Xenotransplantation
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ScienceDirect Journal of Genetics and Genomics xx (2016) 1e5
JGG VIEWS
The Applications of Genome Editing in Xenotransplantation Higher living standards and better medical care are increasing the lifespan of people around the world. Aging populations, however, have an increased incidence of loss of function or failure of cell, tissue or organ. This has led to the development of new medical disciplines, such as organ transplantation and more recently regenerative medicine. Organ transplantation using human donors (allotransplantation) has made enormous progress thanks to the discovery of novel immunosuppressive drugs. However, the growing demand for organs far exceeds the number of organs potentially available from human donors. Xenotransplantation, namely transplantation between animal donors and man, offers the opportunity to use healthy and highly specialized cells, tissues or solid organs readily available for immediate transfer to patients requiring replacement therapy (Ekser et al., 2012). The therapeutic potential of xenotransplantation is wide, some already in clinical use like bioprosthetic heart valves, decellularized pig tissues (skin, ligaments, bone and cartilage), polyclonal antisera, and pancreatic islet, or in a pre-clinical phase like kidney, heart, liver, lung, cornea, and dopaminergic neurons. The pig is a very suitable species for xenotransplantation for reasons that are well documented in the literature, including physiological and anatomical features, and the availability of a high resolution map of the genome. Moreover, the use of pigs for clinical purposes raises little concern from the wider public, because they are already bred by the millions for meat production worldwide. At present, the use of bioprosthetic heart valves of animal origin is a well established xenotransplantation procedure in clinical practice; however, pig islet xenotransplantation has just entered clinical trials (http://www.lctglobal.com/products/ diabecell/about-type-1-diabetes), and life supporting solid organs transplanted into nonhuman primates still do not survive long enough to warrant implementation of clinical trials (Le Bas-Bernardet et al., 2011) although heterotopic heart transplantation in a primate model has resulted in the remarkable survival of almost three years (Mohiuddin et al., 2015). Therefore, several issues still need to be addressed from the safety point of view, and a number of immunological hurdles have been identified (Table 1) and are currently being
addressed at multiple levels (Griesemer et al., 2014). It is expected that the development of novel immunosuppressive strategies for allotransplantation and xenotransplantation, the modification of the immunogenicity of the donor pig through genetic engineering and, possibly the induction of immune tolerance, a phenomenon occasionally observed in allotransplantation, will all contribute to bringing xenotransplantation closer to the clinic (Ekser et al., 2012). All genetically modified pigs for xenotransplantation obtained so far have been generated using somatic cell nuclear transfer (SCNT) technology combined with classical transgenic technology or newly developed genome editing technology in somatic cells. Therefore, SCNT is expected to continue to play a pivotal role to generate founder animals of selected genetic signature for xenotransplantation.
GENOME EDITING FOR XENOTRANSPLANTATION Genetic engineering has been one of the major routes to produce a donor pig whose organs can be tolerated by the human immune system. With the recent discovery and development of programmable nucleases for precise genome modification (Carroll, 2014), the frequency of generating DNA double-strand breaks (DSBs) required for the initiation of genome editing is enhanced by a few logs of times by the precise cutting ability of the programmable nucleases at selected sequences. This technology is expected to have a major impact also on xenotransplantation research. Because of the high targeting efficiency, there is no need of a selectable marker to enrich the targeting events, which makes genetic engineering more “clean” and avoids the integration of undesirable DNA sequences. Amongst the programmable nucleases used today for genome editing, ZFNs (zinc finger nucleases), TALENs (transcription activator-like effector nucleases) and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9), the latter is the most widely used because it is easy to use, flexible and low cost. The full exploitation of genome editing technology requires accurate DNA sequencing information as well as software tools necessary for nuclease design, target site
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Table 1 Immunological barriers to xenotransplantation that can be abrogated through genome editing Problem
Possible cause
Possible target for genome editing
Hyperacute rejection (HAR)
Pre-formed antibodies against Gal and other non-Gal antigens (e.g., Neu5Gc); activation of the complement cascade
GGTA1, CMAH, b4GalNT2, iGb3S and other genes responsible for nonGal antigen production; hCRP (CD55, CD46 and CD59)
Acute humoral xenograft rejection (AHXR)
De novo antibodies against Gal and other non-Gal antigens (e.g., Neu5Gc); activation of the complement cascade; endothelial cell activation; thrombotic microangiopathy; consumptive coagulopathy
hTBM, hEPCR, hA20, TFPI and CD39
Immune cell-mediated rejection (ICMR)
NK and T-cell activation
hTRAIL, CTLA4Ig, HLA-E and hub2m
Instant blood-mediated inflammatory reaction (IBMIR)
Surface proteins; complement activation; innate immunity; activation of platelets and leucocytes
GGTA1, CMAH and b4GalNT2; hCRP (CD55, CD46 and CD59); hTBM, hEPCR, hA20, TFPI and CD39
b4GalNT2, b1,4-N-acetylgalactosaminyltransferase gene; CMAH, cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene; GGTA1, a-1,3galactosyltransferase gene; iGb3S, isogloboside 3 synthase gene; Neu5Gc, N-glycolylneuraminic acid; hCRP, human complement regulatory proteins; hEPCR, human endothelial protein C receptor; TFPI, tissue factor pathway inhibitor; TRAIL, human tumor necrosis factor related apoptosis inducing ligand.
selection and experimental validation (Lee et al., 2016) to avoid undesired side effects in other genomic loci. In general terms, for xenotransplantation applications three types of genetic modifications are required: 1) insertion and expression of a specific desirable transgene of human origin, 2) inactivation or 3) reduced expression of a specific undesired pig gene. Any of these modifications can lead to different effects on the resulting animals, depending on: 1) the site of integration, 2) the number of integrations, and 3) the effects of the genetic modification on the overall homeostasis of the animal. Random integration can disrupt endogenous gene functions required for survival, therefore precise targeting of specific genome sequences with a high frequency through HDR (homology-directed repair) is a must and can only be achieved efficiently with programmable nucleases. The Knock In (KI) of the transgene at a specific location in the pig genome, the so called “safe harbor”, such as ROSA-26 (Li et al., 2014), with the aid of programmable nucleases can control undesirable side-effects, ensure single integration and sustain expression through successive generations without disruption of endogenous functional genes. Moreover, if the transgene, because of its biological activity, is expressed in a tissue-specific (e.g., on endothelial cells or in insulin producing cells) or inducible manner, side-effects of the transgene can be reduced and genome editing can be compatible with the life of the animal. When inactivation of a specific endogenous pig gene is required, genome editing technology can be used for Knock Out (KO) approach via NHEJ (non-homologous end-joining). Major xenoantigens have been readily knocked out by genome editing: the KO of a-1,3-galactosyltransferase (GGTA1) gene encoding the enzyme responsible for the generation of galactose-a-1,3-galactose (Gal) epitope using ZFNs
(Hauschild et al., 2011) or TALENs (Xin et al., 2013), followed by the KO of cytidine monophosphate-Nacetylneuraminic acid hydroxylase (CMAH ) gene coding for the enzyme responsible for the production of N-glycolylneuraminic acid (Neu5Gc) antigen using ZFNs (Kwon et al., 2013; Lutz et al., 2013) and TALENs (Conchon et al., 2013), and more recently by the KO of b1,4-N-acetylgalactosaminyltransferase (b4GalNT2) gene using CRISPR/ Csa9, simultaneously with the KO of GGTA1 and CMAH (Estrada et al., 2015). The third type of genetic modification involves RNA interference technology to reduce gene expression as in the case of pig Tissue Factor (Ahrens et al., 2015), because the KO is not compatible with survival and again the placement of the knockdown vector in a safe harbor with the use of programmable nucleases offers all the advantages already mentioned for the KI. GENOME EDITING AND SAFETY Questions regarding safety have beset the xenotransplantation field for almost 20 years, mainly because of the discovery that porcine endogenous retroviruses (PERVs) could infect human cells in vitro (Patience et al., 1997). PERVs are integrated in the pig genome in very variable copy numbers (up to more than 100 copies/genome) and generate different levels of circulating RNA. Several lines of investigation have convincingly shown that PERVs are not transmitted to patients receiving living pig xenografts (Scobie et al., 2013). Hence, the current understanding is that the risks might have been previously overestimated. Nevertheless, means of reducing PERV mRNAs have been devised by selecting specific pigs that are naturally PERV C negative, being PERV C the most
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dangerous type, and that have low copies of PERV A and B. An ultimate genome editing approach has been reported by Yang et al. (2015) with the use of the CRISPR/Cas9 to target and inactivate the pol gene in all the 62 copies of integrated PERVs in a PK15 immortalized pig cell line. In that study, Cas9 was constitutively expressed under an inducible promoter in PK15 cells for two weeks to achieve high targeting efficiency. Inspite of this impressive genome editing result, it is not known whether such a massive genome editing for a prolonged period of time and the complete KO of all PERVs will be compatible with generating a viable pig, since endogenous retroviruses are highly expressed in placenta of different species and recently their physiological functions during gestation has been reviewed by Denner (2016). In humans, Syncytin1 and Syncytin2 are highly conserved retroviral envelope proteins and are involved in placentogenesis due to their fusogenic and immunosuppressive properties (Venables et al., 1995). Across the eutherian mammals studied, even if different placental structures are described, Syncytin-like proteins are expressed in the placenta with similar functions. The only known exceptions are pigs, characterized by epitheliochorial placenta, where Syncytin-like proteins have not been described (Denner, 2016). Nonetheless, for more conventional transmissible infectious agents, classical control measures based on defining pathogen free herds can be implemented and can be effective (Fishman et al., 2012). Finally, genome editing technology could also help in making animals resistant to certain pathogens by genome editing-mediated expression of RNA sequences complementary to viral mRNA, which will result in RNA interference. REGULATORY ASPECTS Xenotransplantation is a rather young field of medicine, yet it has a potentially significant impact on the welfare and health of the general population. The first concern of regulatory authorities has been the safety aspects related to the use of animals for clinical purposes, and guidelines have been issued by several regulatory agencies. Furthermore, the World Health Organization (WHO) has previously stated its position on the subject in the context of the 57th World Health Assembly in Resolution WHA57.18 (http://www.transplant-observatory. org/SiteCollectionDocuments/wha57resen.pdf). However, xenotransplantation requires an additional level of safety because pig donors will mainly be genetically modified, a factor that the regulatory agencies will have to take into account. The Food and Drug Administration (FDA) has already issued recommendations for the regulatory framework of genetically engineered animals containing heritable recombinant DNA vectors (http://www.fda.gov/ForConsumers/ ConsumerUpdates/ucm048106.htm). Drugs produced in transgenic animals have been already approved for clinical use in patients, such as ATryn produced by transgenic goats and approved by both the European Medicines Agency (EMA) and FDA, and Ruconest produced by transgenic rabbits and approved by both EMA and FDA. For this reason, it is the
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authors’ perception that the basic regulatory framework is already in place and this can readily be adapted to include the specific case of xenotransplantation. Therefore, it is expected that genetic engineering in general and genome editing in particular will not represent an insurmountable obstacle to the approval of possible xenotransplantation products once clinical efficacy has been convincingly demonstrated.
FUTURE PERSPECTIVES The successful clinical application of xenotransplantation as described above will depend on several factors, and genetic engineering is one of the most important. Indeed, in combination with genetic engineering, the development of novel immunosuppressive strategies that may enable the induction of immunotolerance remains important research areas where advances are awaited. As far as genetic engineering is concerned, as previously illustrated, enormous progress has been made in generating large animals carrying the required genetic modifications. Progress has been made regarding the efficiency, precision and multiplexing of genetic modifications. Instrumental has been the advent of SCNT, because donor cells are selected in advance after genetic engineering and all SCNT-generated animals carry the expected genetic modifications. Moreover, because it is expected that multiplex genome editing intervention will be required and will have to be stacked in the same pig genome, SCNT will continue to play a pivotal role for xenotransplantation. The use of programmable nucleases (ZFNs, TALENs and CRISPR/Cas9) has made the procedures for gene targeting extremely precise and effective. As a consequence, any given gene whose sequence is known can be inactivated in a matter of weeks. The integration of a new gene into the genome can be directed to “safe harbor” sites to obtain a single integration without disrupting endogenous genes and ensure functional activity throughout breeding of successive generations. Although genome editing can provide unprecedented achievements, there will be a limit to the number of edited genes that will be compatible with a functional pig genome. The regulatory agencies and the stakeholders involved are considering whether the animals generated by genome editing technology should be classified or not as Genetically Modified Organisms. Indeed, if only the indel (insertion and deletion) mutations are introduced in the genome with no trace of foreign DNA, the resulting animals are not different from spontaneously occurring mutants; therefore, at least in theory, there should be no requirement for a specific regulatory framework. Because of the high efficacy of programmable nucleases in inducing DSBs in the genome, pig zygote microinjection (Whitworth et al., 2014) is currently being explored as a route to the direct generation of living genome-edited animals for some applications where the genetic background of the pig line is not important or simple proofs of concept are required to test the effects of a single new molecule in xenotransplantation.
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Another route, which is being actively explored to overcome immune rejection to bring xenotransplantation closer to the clinic, is aimed at generating pig-human chimeric organs. If a pig organ is made up of human cells, the human immune system should better tolerate it. In this scenario, defective pig embryos for one target organ can be generated by genetic engineering technology and then aggregated with pluripotent stem cells (PSCs) of human origin (blastocyst complementation), and thus during the development of the resulting animal the defective organ will be generated by the PSCs. For example, Pdx1 regulates pancreas development and during development the genetically modified Pdx1/ embryos do not form the pancreas. When the defective Pdx1/ mouse embryos are aggregated with rat Pdx1þ/þ PSCs, the resulting pancreas is entirely derived from rat Pdx1þ/þ PSCs (Kobayashi et al., 2010). A similar proof of principle experiment has been carried out in the pig (Matsunari et al., 2013), resulting in the generation of pigs with the pancreas derived from PSCs used for blastocyst complementation. Sall1 is a gene controlling kidney development, and KO of this gene in the mouse generates pups lacking all kidney parenchyma except for collecting ducts and microvasculature. When defective Sall1/ blastocysts are complemented with normal mouse-derived PSCs, normal kidney development is restored and most of the organ derives from the PSCs (Usui et al., 2012). In the mouse experiment described above, defective embryos were generated by breeding heterozygous mutant animals obtained by complex conventional genetic engineering approaches and stem cell technology. This route is not
practical for a large animal like the pig. Therefore, generation of defective pig embryos now will benefit from the combinational use of programmable nucleases and SCNT. The genome of somatic cells can be edited by programmable nucleases during culture to generate the KO of the relevant master gene responsible for a given organ. The defective embryo is then generated by SCNT, and as such it will generate a non-viable animal; however, when the embryo is complemented with PSCs, the defective organ will be restored and the animal will be viable. Future experiments will be directed to the use of human induced pluripotent stem cells (iPSCs) to be aggregated with defective pig embryos to generate pigs carrying specific organs primarily derived from human cells (Fig. 1). The generation of pig-human interspecific chimeras will inevitably raise ethical concerns for possible contribution of human iPSCs to the pig brain. These concerns could also be addressed by genome editing of the human iPSCs for the master genes responsible for central nervous system (CNS) development, thus emphasizing once again the central role of programmable nucleases. Given the number and the complexity of the diseases that could be potentially treated with xenotransplantation, it is likely that there will be different solutions as to whether entire organs, tissues or cells will be required. Furthermore, it is unquestionable that the newly developed genome editing technology will be a key tool in such developments; especially it has become efficient, effective and affordable in many laboratories around the world.
Human iPSCs disabled for CNS genes by genome editing
Human iPSC injection into pig blastocysts genetically modified by genome editing to lack a selected organ (e.g.,kidney)
Patient receiving human kidney developed in a genome edited pig
Pig with kidney essentially derived from human iPSCs Fig. 1. Concept and application of chimeric organ complementation for xenotransplantation. Induced pluripotent stem cells (iPSCs) derived from patients can be edited to disable central nervous system (CNS) development. Pig somatic cells or zygotes can be edited by CRISPR/Cas9 to disable selected genes responsible for organ specification (kidney, pancreas, etc.). Specific organ-disabled pig embryos are aggregated with human patient-derived iPSCs. The resulting chimeric pig will have the organ formed by and large of human iPSCs. Please cite this article in press as: Perota, A., et al., The Applications of Genome Editing in Xenotransplantation, Journal of Genetics and Genomics (2016), http://dx.doi.org/10.1016/j.jgg.2016.04.012
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ACKNOWLEDGMENTS This article concerns part of the work carried out under the European Union Seventh Framework Programme collaborative Projects Translink (Grant agreement No. 603049) and Xenoislet (Grant agreement No. 601827) as well as the ERC project MitCare (Grant agreement No. 322424).
Andrea Perotaa, Irina Lagutinaa, Corinne Quadaltia,b, Giovanna Lazzaria,c, Emanuele Cozzid, Cesare Gallia,b,c,* a
Avantea, Laboratory of Reproductive Technologies, Cremona, Italy
b
Department of Veterinary Medical Sciences University of Bologna, Italy c
Avantea Foundation, Cremona, Italy
d
Transplant Immunology Unit, Padua General Hospital, Padua, Italy *Corresponding author. Tel: þ39 0372 43 7242. E-mail address: [email protected] (C. Galli)
Received 22 April 2016 Accepted 28 April 2016 Available online xxx
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Kobayashi, T., Yamaguchi, T., Hamanaka, S., Kato-Itoh, M., Yamazaki, Y., Ibata, M., Sato, H., Lee, Y.S., Usui, J., Knisely, A.S., Hirabayashi, M., Nakauchi, H., 2010. Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells. Cell 142, 787e799. Le Bas-Bernardet, S., Tillou, X., Poirier, N., Dilek, N., Chatelais, M., Devalliere, J., Charreau, B., Minault, D., Hervouet, J., Renaudin, K., Crossan, C., Scobie, L., Cowan, P.J., d’Apice, A.J., Galli, C., Cozzi, E., Soulillou, J.P., Vanhove, B., Blancho, G., 2011. Xenotransplantation of galactosyl-transferase knockout, CD55, CD59, CD39, and fucosyltransferase transgenic pig kidneys into baboons. Transplant. Proc. 43, 3426e3430. Lee, C.M., Cradick, T.J., Fine, E.J., Bao, G., 2016. Nuclease target site selection for maximizing on-target activity and minimizing off-target effects in genome editing. Mol. Ther. 24, 475e487. Li, X., Yang, Y., Bu, L., Guo, X., Tang, C., Song, J., Fan, N., Zhao, B., Ouyang, Z., Liu, Z., Zhao, Y., Yi, X., Quan, L., Liu, S., Yang, Z., Ouyang, H., Chen, Y.E., Wang, Z., Lai, L., 2014. Rosa26-targeted swine models for stable gene over-expression and Cre-mediated lineage tracing. Cell Res. 24, 501e504. Lutz, A.J., Li, P., Estrada, J.L., Sidner, R.A., Chihara, R.K., Downey, S.M., Burlak, C., Wang, Z.Y., Reyes, L.M., Ivary, B., Yin, F., Blankenship, R.L., Paris, L.L., Tector, A.J., 2013. Double knockout pigs deficient in N-glycolylneuraminic acid and galactose alpha-1,3-galactose reduce the humoral barrier to xenotransplantation. Xenotransplantation 20, 27e35. Matsunari, H., Nagashima, H., Watanabe, M., Umeyama, K., Nakano, K., Nagaya, M., Kobayashi, T., Yamaguchi, T., Sumazaki, R., Herzenberg, L.A., Nakauchi, H., 2013. Blastocyst complementation generates exogenic pancreas in vivo in apancreatic cloned pigs. Proc. Natl. Acad. Sci. USA 110, 4557e4562. Mohiuddin, M., Singh, A., Corcoran, P., Thomas, M., Lewis, B., Clark, T., Eckhaus, M., Belli, A., Reimann, K., Klymuik, N., Wolf, E., Ayares, D., K, H., 2015. Critical need of continuous co-stimulation blockade with anti CD40 antibody (2C10.R4) for long-term maintenance of GTKO.hCD46.hTBM pig cardiac xenograft survival in baboons. In: Xenotransplantation, Abstracts of the IPITA-IXA-CTS 2015 Joint Congress November 15e19, 2015, Melbourne, Australia, vol. 22, pp. S121eS184. Patience, C., Takeuchi, Y., Weiss, R.A., 1997. Infection of human cells by an endogenous retrovirus of pigs. Nat. Med. 3, 282e286. Scobie, L., Padler-Karavani, V., Le Bas-Bernardet, S., Crossan, C., Blaha, J., Matouskova, M., Hector, R.D., Cozzi, E., Vanhove, B., Charreau, B., Blancho, G., Bourdais, L., Tallacchini, M., Ribes, J.M., Yu, H., Chen, X., Kracikova, J., Broz, L., Hejnar, J., Vesely, P., Takeuchi, Y., Varki, A., Soulillou, J.P., 2013. Long-term IgG response to porcine Neu5Gc antigens without transmission of PERV in burn patients treated with porcine skin xenografts. J. Immunol. 191, 2907e2915. Usui, J., Kobayashi, T., Yamaguchi, T., Knisely, A.S., Nishinakamura, R., Nakauchi, H., 2012. Generation of kidney from pluripotent stem cells via blastocyst complementation. Am. J. Pathol. 180, 2417e2426. Venables, P.J., Brookes, S.M., Griffiths, D., Weiss, R.A., Boyd, M.T., 1995. Abundance of an endogenous retroviral envelope protein in placental trophoblasts suggests a biological function. Virology 211, 589e592. Whitworth, K.M., Lee, K., Benne, J.A., Beaton, B.P., Spate, L.D., Murphy, S.L., Samuel, M.S., Mao, J., O’Gorman, C., Walters, E.M., Murphy, C.N., Driver, J., Mileham, A., McLaren, D., Wells, K.D., Prather, R.S., 2014. Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos. Biol. Reprod. 91, 78. Xin, J., Yang, H., Fan, N., Zhao, B., Ouyang, Z., Liu, Z., Zhao, Y., Li, X., Song, J., Yang, Y., Zou, Q., Yan, Q., Zeng, Y., Lai, L., 2013. Highly efficient generation of GGTA1 biallelic knockout inbred mini-pigs with TALENs. PLoS One 8, e84250. Yang, L., Guell, M., Niu, D., George, H., Lesha, E., Grishin, D., Aach, J., Shrock, E., Xu, W., Poci, J., Cortazio, R., Wilkinson, R.A., Fishman, J.A., Church, G., 2015. Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science 350, 1101e1104.
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ScienceDirect Journal of Genetics and Genomics xx (2016) 1e5
JGG VIEWS
The Applications of Genome Editing in Xenotransplantation Higher living standards and better medical care are increasing the lifespan of people around the world. Aging populations, however, have an increased incidence of loss of function or failure of cell, tissue or organ. This has led to the development of new medical disciplines, such as organ transplantation and more recently regenerative medicine. Organ transplantation using human donors (allotransplantation) has made enormous progress thanks to the discovery of novel immunosuppressive drugs. However, the growing demand for organs far exceeds the number of organs potentially available from human donors. Xenotransplantation, namely transplantation between animal donors and man, offers the opportunity to use healthy and highly specialized cells, tissues or solid organs readily available for immediate transfer to patients requiring replacement therapy (Ekser et al., 2012). The therapeutic potential of xenotransplantation is wide, some already in clinical use like bioprosthetic heart valves, decellularized pig tissues (skin, ligaments, bone and cartilage), polyclonal antisera, and pancreatic islet, or in a pre-clinical phase like kidney, heart, liver, lung, cornea, and dopaminergic neurons. The pig is a very suitable species for xenotransplantation for reasons that are well documented in the literature, including physiological and anatomical features, and the availability of a high resolution map of the genome. Moreover, the use of pigs for clinical purposes raises little concern from the wider public, because they are already bred by the millions for meat production worldwide. At present, the use of bioprosthetic heart valves of animal origin is a well established xenotransplantation procedure in clinical practice; however, pig islet xenotransplantation has just entered clinical trials (http://www.lctglobal.com/products/ diabecell/about-type-1-diabetes), and life supporting solid organs transplanted into nonhuman primates still do not survive long enough to warrant implementation of clinical trials (Le Bas-Bernardet et al., 2011) although heterotopic heart transplantation in a primate model has resulted in the remarkable survival of almost three years (Mohiuddin et al., 2015). Therefore, several issues still need to be addressed from the safety point of view, and a number of immunological hurdles have been identified (Table 1) and are currently being
addressed at multiple levels (Griesemer et al., 2014). It is expected that the development of novel immunosuppressive strategies for allotransplantation and xenotransplantation, the modification of the immunogenicity of the donor pig through genetic engineering and, possibly the induction of immune tolerance, a phenomenon occasionally observed in allotransplantation, will all contribute to bringing xenotransplantation closer to the clinic (Ekser et al., 2012). All genetically modified pigs for xenotransplantation obtained so far have been generated using somatic cell nuclear transfer (SCNT) technology combined with classical transgenic technology or newly developed genome editing technology in somatic cells. Therefore, SCNT is expected to continue to play a pivotal role to generate founder animals of selected genetic signature for xenotransplantation.
GENOME EDITING FOR XENOTRANSPLANTATION Genetic engineering has been one of the major routes to produce a donor pig whose organs can be tolerated by the human immune system. With the recent discovery and development of programmable nucleases for precise genome modification (Carroll, 2014), the frequency of generating DNA double-strand breaks (DSBs) required for the initiation of genome editing is enhanced by a few logs of times by the precise cutting ability of the programmable nucleases at selected sequences. This technology is expected to have a major impact also on xenotransplantation research. Because of the high targeting efficiency, there is no need of a selectable marker to enrich the targeting events, which makes genetic engineering more “clean” and avoids the integration of undesirable DNA sequences. Amongst the programmable nucleases used today for genome editing, ZFNs (zinc finger nucleases), TALENs (transcription activator-like effector nucleases) and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9), the latter is the most widely used because it is easy to use, flexible and low cost. The full exploitation of genome editing technology requires accurate DNA sequencing information as well as software tools necessary for nuclease design, target site
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Table 1 Immunological barriers to xenotransplantation that can be abrogated through genome editing Problem
Possible cause
Possible target for genome editing
Hyperacute rejection (HAR)
Pre-formed antibodies against Gal and other non-Gal antigens (e.g., Neu5Gc); activation of the complement cascade
GGTA1, CMAH, b4GalNT2, iGb3S and other genes responsible for nonGal antigen production; hCRP (CD55, CD46 and CD59)
Acute humoral xenograft rejection (AHXR)
De novo antibodies against Gal and other non-Gal antigens (e.g., Neu5Gc); activation of the complement cascade; endothelial cell activation; thrombotic microangiopathy; consumptive coagulopathy
hTBM, hEPCR, hA20, TFPI and CD39
Immune cell-mediated rejection (ICMR)
NK and T-cell activation
hTRAIL, CTLA4Ig, HLA-E and hub2m
Instant blood-mediated inflammatory reaction (IBMIR)
Surface proteins; complement activation; innate immunity; activation of platelets and leucocytes
GGTA1, CMAH and b4GalNT2; hCRP (CD55, CD46 and CD59); hTBM, hEPCR, hA20, TFPI and CD39
b4GalNT2, b1,4-N-acetylgalactosaminyltransferase gene; CMAH, cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene; GGTA1, a-1,3galactosyltransferase gene; iGb3S, isogloboside 3 synthase gene; Neu5Gc, N-glycolylneuraminic acid; hCRP, human complement regulatory proteins; hEPCR, human endothelial protein C receptor; TFPI, tissue factor pathway inhibitor; TRAIL, human tumor necrosis factor related apoptosis inducing ligand.
selection and experimental validation (Lee et al., 2016) to avoid undesired side effects in other genomic loci. In general terms, for xenotransplantation applications three types of genetic modifications are required: 1) insertion and expression of a specific desirable transgene of human origin, 2) inactivation or 3) reduced expression of a specific undesired pig gene. Any of these modifications can lead to different effects on the resulting animals, depending on: 1) the site of integration, 2) the number of integrations, and 3) the effects of the genetic modification on the overall homeostasis of the animal. Random integration can disrupt endogenous gene functions required for survival, therefore precise targeting of specific genome sequences with a high frequency through HDR (homology-directed repair) is a must and can only be achieved efficiently with programmable nucleases. The Knock In (KI) of the transgene at a specific location in the pig genome, the so called “safe harbor”, such as ROSA-26 (Li et al., 2014), with the aid of programmable nucleases can control undesirable side-effects, ensure single integration and sustain expression through successive generations without disruption of endogenous functional genes. Moreover, if the transgene, because of its biological activity, is expressed in a tissue-specific (e.g., on endothelial cells or in insulin producing cells) or inducible manner, side-effects of the transgene can be reduced and genome editing can be compatible with the life of the animal. When inactivation of a specific endogenous pig gene is required, genome editing technology can be used for Knock Out (KO) approach via NHEJ (non-homologous end-joining). Major xenoantigens have been readily knocked out by genome editing: the KO of a-1,3-galactosyltransferase (GGTA1) gene encoding the enzyme responsible for the generation of galactose-a-1,3-galactose (Gal) epitope using ZFNs
(Hauschild et al., 2011) or TALENs (Xin et al., 2013), followed by the KO of cytidine monophosphate-Nacetylneuraminic acid hydroxylase (CMAH ) gene coding for the enzyme responsible for the production of N-glycolylneuraminic acid (Neu5Gc) antigen using ZFNs (Kwon et al., 2013; Lutz et al., 2013) and TALENs (Conchon et al., 2013), and more recently by the KO of b1,4-N-acetylgalactosaminyltransferase (b4GalNT2) gene using CRISPR/ Csa9, simultaneously with the KO of GGTA1 and CMAH (Estrada et al., 2015). The third type of genetic modification involves RNA interference technology to reduce gene expression as in the case of pig Tissue Factor (Ahrens et al., 2015), because the KO is not compatible with survival and again the placement of the knockdown vector in a safe harbor with the use of programmable nucleases offers all the advantages already mentioned for the KI. GENOME EDITING AND SAFETY Questions regarding safety have beset the xenotransplantation field for almost 20 years, mainly because of the discovery that porcine endogenous retroviruses (PERVs) could infect human cells in vitro (Patience et al., 1997). PERVs are integrated in the pig genome in very variable copy numbers (up to more than 100 copies/genome) and generate different levels of circulating RNA. Several lines of investigation have convincingly shown that PERVs are not transmitted to patients receiving living pig xenografts (Scobie et al., 2013). Hence, the current understanding is that the risks might have been previously overestimated. Nevertheless, means of reducing PERV mRNAs have been devised by selecting specific pigs that are naturally PERV C negative, being PERV C the most
Please cite this article in press as: Perota, A., et al., The Applications of Genome Editing in Xenotransplantation, Journal of Genetics and Genomics (2016), http://dx.doi.org/10.1016/j.jgg.2016.04.012
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dangerous type, and that have low copies of PERV A and B. An ultimate genome editing approach has been reported by Yang et al. (2015) with the use of the CRISPR/Cas9 to target and inactivate the pol gene in all the 62 copies of integrated PERVs in a PK15 immortalized pig cell line. In that study, Cas9 was constitutively expressed under an inducible promoter in PK15 cells for two weeks to achieve high targeting efficiency. Inspite of this impressive genome editing result, it is not known whether such a massive genome editing for a prolonged period of time and the complete KO of all PERVs will be compatible with generating a viable pig, since endogenous retroviruses are highly expressed in placenta of different species and recently their physiological functions during gestation has been reviewed by Denner (2016). In humans, Syncytin1 and Syncytin2 are highly conserved retroviral envelope proteins and are involved in placentogenesis due to their fusogenic and immunosuppressive properties (Venables et al., 1995). Across the eutherian mammals studied, even if different placental structures are described, Syncytin-like proteins are expressed in the placenta with similar functions. The only known exceptions are pigs, characterized by epitheliochorial placenta, where Syncytin-like proteins have not been described (Denner, 2016). Nonetheless, for more conventional transmissible infectious agents, classical control measures based on defining pathogen free herds can be implemented and can be effective (Fishman et al., 2012). Finally, genome editing technology could also help in making animals resistant to certain pathogens by genome editing-mediated expression of RNA sequences complementary to viral mRNA, which will result in RNA interference. REGULATORY ASPECTS Xenotransplantation is a rather young field of medicine, yet it has a potentially significant impact on the welfare and health of the general population. The first concern of regulatory authorities has been the safety aspects related to the use of animals for clinical purposes, and guidelines have been issued by several regulatory agencies. Furthermore, the World Health Organization (WHO) has previously stated its position on the subject in the context of the 57th World Health Assembly in Resolution WHA57.18 (http://www.transplant-observatory. org/SiteCollectionDocuments/wha57resen.pdf). However, xenotransplantation requires an additional level of safety because pig donors will mainly be genetically modified, a factor that the regulatory agencies will have to take into account. The Food and Drug Administration (FDA) has already issued recommendations for the regulatory framework of genetically engineered animals containing heritable recombinant DNA vectors (http://www.fda.gov/ForConsumers/ ConsumerUpdates/ucm048106.htm). Drugs produced in transgenic animals have been already approved for clinical use in patients, such as ATryn produced by transgenic goats and approved by both the European Medicines Agency (EMA) and FDA, and Ruconest produced by transgenic rabbits and approved by both EMA and FDA. For this reason, it is the
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authors’ perception that the basic regulatory framework is already in place and this can readily be adapted to include the specific case of xenotransplantation. Therefore, it is expected that genetic engineering in general and genome editing in particular will not represent an insurmountable obstacle to the approval of possible xenotransplantation products once clinical efficacy has been convincingly demonstrated.
FUTURE PERSPECTIVES The successful clinical application of xenotransplantation as described above will depend on several factors, and genetic engineering is one of the most important. Indeed, in combination with genetic engineering, the development of novel immunosuppressive strategies that may enable the induction of immunotolerance remains important research areas where advances are awaited. As far as genetic engineering is concerned, as previously illustrated, enormous progress has been made in generating large animals carrying the required genetic modifications. Progress has been made regarding the efficiency, precision and multiplexing of genetic modifications. Instrumental has been the advent of SCNT, because donor cells are selected in advance after genetic engineering and all SCNT-generated animals carry the expected genetic modifications. Moreover, because it is expected that multiplex genome editing intervention will be required and will have to be stacked in the same pig genome, SCNT will continue to play a pivotal role for xenotransplantation. The use of programmable nucleases (ZFNs, TALENs and CRISPR/Cas9) has made the procedures for gene targeting extremely precise and effective. As a consequence, any given gene whose sequence is known can be inactivated in a matter of weeks. The integration of a new gene into the genome can be directed to “safe harbor” sites to obtain a single integration without disrupting endogenous genes and ensure functional activity throughout breeding of successive generations. Although genome editing can provide unprecedented achievements, there will be a limit to the number of edited genes that will be compatible with a functional pig genome. The regulatory agencies and the stakeholders involved are considering whether the animals generated by genome editing technology should be classified or not as Genetically Modified Organisms. Indeed, if only the indel (insertion and deletion) mutations are introduced in the genome with no trace of foreign DNA, the resulting animals are not different from spontaneously occurring mutants; therefore, at least in theory, there should be no requirement for a specific regulatory framework. Because of the high efficacy of programmable nucleases in inducing DSBs in the genome, pig zygote microinjection (Whitworth et al., 2014) is currently being explored as a route to the direct generation of living genome-edited animals for some applications where the genetic background of the pig line is not important or simple proofs of concept are required to test the effects of a single new molecule in xenotransplantation.
Please cite this article in press as: Perota, A., et al., The Applications of Genome Editing in Xenotransplantation, Journal of Genetics and Genomics (2016), http://dx.doi.org/10.1016/j.jgg.2016.04.012
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Another route, which is being actively explored to overcome immune rejection to bring xenotransplantation closer to the clinic, is aimed at generating pig-human chimeric organs. If a pig organ is made up of human cells, the human immune system should better tolerate it. In this scenario, defective pig embryos for one target organ can be generated by genetic engineering technology and then aggregated with pluripotent stem cells (PSCs) of human origin (blastocyst complementation), and thus during the development of the resulting animal the defective organ will be generated by the PSCs. For example, Pdx1 regulates pancreas development and during development the genetically modified Pdx1/ embryos do not form the pancreas. When the defective Pdx1/ mouse embryos are aggregated with rat Pdx1þ/þ PSCs, the resulting pancreas is entirely derived from rat Pdx1þ/þ PSCs (Kobayashi et al., 2010). A similar proof of principle experiment has been carried out in the pig (Matsunari et al., 2013), resulting in the generation of pigs with the pancreas derived from PSCs used for blastocyst complementation. Sall1 is a gene controlling kidney development, and KO of this gene in the mouse generates pups lacking all kidney parenchyma except for collecting ducts and microvasculature. When defective Sall1/ blastocysts are complemented with normal mouse-derived PSCs, normal kidney development is restored and most of the organ derives from the PSCs (Usui et al., 2012). In the mouse experiment described above, defective embryos were generated by breeding heterozygous mutant animals obtained by complex conventional genetic engineering approaches and stem cell technology. This route is not
practical for a large animal like the pig. Therefore, generation of defective pig embryos now will benefit from the combinational use of programmable nucleases and SCNT. The genome of somatic cells can be edited by programmable nucleases during culture to generate the KO of the relevant master gene responsible for a given organ. The defective embryo is then generated by SCNT, and as such it will generate a non-viable animal; however, when the embryo is complemented with PSCs, the defective organ will be restored and the animal will be viable. Future experiments will be directed to the use of human induced pluripotent stem cells (iPSCs) to be aggregated with defective pig embryos to generate pigs carrying specific organs primarily derived from human cells (Fig. 1). The generation of pig-human interspecific chimeras will inevitably raise ethical concerns for possible contribution of human iPSCs to the pig brain. These concerns could also be addressed by genome editing of the human iPSCs for the master genes responsible for central nervous system (CNS) development, thus emphasizing once again the central role of programmable nucleases. Given the number and the complexity of the diseases that could be potentially treated with xenotransplantation, it is likely that there will be different solutions as to whether entire organs, tissues or cells will be required. Furthermore, it is unquestionable that the newly developed genome editing technology will be a key tool in such developments; especially it has become efficient, effective and affordable in many laboratories around the world.
Human iPSCs disabled for CNS genes by genome editing
Human iPSC injection into pig blastocysts genetically modified by genome editing to lack a selected organ (e.g.,kidney)
Patient receiving human kidney developed in a genome edited pig
Pig with kidney essentially derived from human iPSCs Fig. 1. Concept and application of chimeric organ complementation for xenotransplantation. Induced pluripotent stem cells (iPSCs) derived from patients can be edited to disable central nervous system (CNS) development. Pig somatic cells or zygotes can be edited by CRISPR/Cas9 to disable selected genes responsible for organ specification (kidney, pancreas, etc.). Specific organ-disabled pig embryos are aggregated with human patient-derived iPSCs. The resulting chimeric pig will have the organ formed by and large of human iPSCs. Please cite this article in press as: Perota, A., et al., The Applications of Genome Editing in Xenotransplantation, Journal of Genetics and Genomics (2016), http://dx.doi.org/10.1016/j.jgg.2016.04.012
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ACKNOWLEDGMENTS This article concerns part of the work carried out under the European Union Seventh Framework Programme collaborative Projects Translink (Grant agreement No. 603049) and Xenoislet (Grant agreement No. 601827) as well as the ERC project MitCare (Grant agreement No. 322424).
Andrea Perotaa, Irina Lagutinaa, Corinne Quadaltia,b, Giovanna Lazzaria,c, Emanuele Cozzid, Cesare Gallia,b,c,* a
Avantea, Laboratory of Reproductive Technologies, Cremona, Italy
b
Department of Veterinary Medical Sciences University of Bologna, Italy c
Avantea Foundation, Cremona, Italy
d
Transplant Immunology Unit, Padua General Hospital, Padua, Italy *Corresponding author. Tel: þ39 0372 43 7242. E-mail address: [email protected] (C. Galli)
Received 22 April 2016 Accepted 28 April 2016 Available online xxx
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Please cite this article in press as: Perota, A., et al., The Applications of Genome Editing in Xenotransplantation, Journal of Genetics and Genomics (2016), http://dx.doi.org/10.1016/j.jgg.2016.04.012