Xenotransplantation

Transgenic pigs are considered to be a perfect organ donor for humans to reduce or even eliminate the growing shortage of suitable human organs (COOPER and AYARES 2011). Due to the lack of suitable organs for allotransplantation many people die while waiting for a human donor organ (an average of three per day in Germany). In Germany about 8,000 dialyze-patients are waiting for a kidney. In 2011, 2,850 patients were transplanted, while the number of required organs was three times higher, resulting in a waiting time for a kidney of 5-6 years (Figure 1) (DSO 2012).

Figure 1: Waiting list and number of kidney transplantations in Germany from 2002 to 2011 (DSO 2012)

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The use of concordant xenografts (between closely related species) like primate-to-human transplantation would lead to an immunological response similar to allotransplantation, which can be controlled by an efficient immunosuppressive treatment.

High costs, the difficulty to breed and keep the animals under strict hygienic conditions, the risk of zoonosis and ethical concerns have prevented primates to be used as organ donors for human patients. Additionally, most of the potential primate species are already on the list of endangered species. After discordant xenotransplantation (between distantly related species) a more severe immune response appears which can not be prevented by immunosuppressive agents. The domestic pig is considered as perfect organ donor for humans, due to its physiological similarity, size-matching organs, low production costs, high reproductive capacity and the knowledge about maintaining pigs under hygienic specific-pathogen-free conditions (PETERSEN et al. 2009a).

Transplantation of discordant xenografts leads to hyperacute rejection (HAR) within minutes after transplantation and finally destroys the transplanted organ. The responsible major antigens are carbohydrate structures called Gal-epitopes on the surface of porcine cells and tissues (Figure 2). The α1,3-galactosyltransferase encoded by the GGTA1 gene synthesizes these epitopes on porcine cells. Gal-epitopes are attacked by the human immune system immediately after transplantations. About 1% of all circulating antibodies in the human blood are directed against Gal-epitopes. Binding of pre-formed antibodies activates the complement cascade, that ultimately leads to the formation of a membrane attack complex (MAC) which in turn causes cell lysis and xenograft rejection (PARKER et al.

1996).

The presence of Gal-epitopes in placental and marsupial mammals, but not in non-mammalian vertebrates indicates the evolutionary appearance of the GGTA1 gene about 80 million years ago (GALILI 1995). Human and old world monkeys lost the function of this gene about 28 million years ago. It might have been an evolutionary advantage to express anti-Gal antibodies for ancestral old world higher primates, which were exposed to endemic pathogens (viruses, bacteria and protozoa possess Gal-epitopes). One requirement for the production of anti-Gal antibodies was the suppression of Gal-epitopes by a naturally

occurring gene knockout in order to prevent autoimmune response (GALILI 1995; GALILI and SWANSON 1991).

GGTA1-KO pigs were produced by using conventional genetic modification tools (DAI et al. 2002; PHELPS et al. 2003). Transplantation of kidneys and hearts from pigs with a homozygous GGTA1-KO to baboons with support of immunosuppressive agents resulted in significantly improved organ survival up to 179 days (KUWAKI et al. 2005; TSENG et al. 2005;

YAMADA et al. 2005). The production of transgenic pigs is essential for studies in the field of xenotransplantation. Table 3 summarizes published achievements in pig-to-baboon Figure 2: Hyperacute rejection (HAR) after anti-Gal antibodies bind to Gal-epitopes. (A) Binding of anti-Gal antibodies to Gal-epitopes induces complement cascade activation, resulting in a membrane attack complex (MAC), which is responsible for movement of ions and water across the membrane and ultimately for cell lysis; Decay accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and membrane inhibitor of reactive lysis (MIRL, CD59) can prevent the formation of MAC; C3-C9 are transition proteins responsible for the formations of MACs (modified from TURNBERG and BOTTO 2003). (B) Humoral graft rejection; Binding of preformed anti-Gal antibodies to endothelial cells of a xenograft leads to complement activation and formation of MACs and finally to massive haemorrhage and thrombosis (modified from YANG and SYKES 2007).

(TURNBERG and BOTTO 2003) (YANG and SYKES 2007)

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xenotransplantation. To further prolong survival of xenografts, multi-transgenic pigs are required to overcome the hurdle of HAR.

Various attempts have been made to prevent rejection of porcine xenografts.

Complement regulatory proteins such as human decay accelerating factor (hDAF, or hCD55) work in a species specific manner. Organs from transgenic animals expressing hCD55 were compatible with reduced complement-mediated injury (Figure 2). Transgenic pig hearts expressing hCD55 were transplanted into baboons, and led to a survival for up to 99 days (BHATTI et al. 1999). The combination of Gal-KO and hCD55 transgenic pig in xenotransplantation did not improve graft survival, but hCD55 expression seemed to restrict local complement activation (McGREGOR et al. 2012). Expression of hCD59 (membrane inhibitor of reactive lysis, MIRL) on islet cells led to reduced complement-mediated lysis (SCHMIDT et al. 2003). HCD46 transgenic pigs were generated and increased number of MCP proteins lead to enhanced protection of host cells from damage by complement activation (McGREGOR et al. 2005). A combination of Gal-KO, hCD46-KI and B-cell depletion showed a maximum xenograft survival of 236 days in regard of entire organs (MOHIUDDIN et al. 2012).

(MCGREGOR et al. 2012)

Table 3: Summary of pig-to-baboon transplantation regarding genetically knockin (KI) or knockout (KO), kind of organ and maximum survival (days)

Type of genetic engineering

Organ Max. survival

(days)

Authors

CD59-KI and hCD55-KI Heart 30h McCURRY et al. 1995

Gal-KO Kidney 16 CHEN et al. 2005

Gal-KO and hCD55-KI Heart, heterotopic 28* McGREGOR et al. 2012

hCD55-KI Heart, heterotopic 39 VIAL et al. 2000

hCD55-KI Kidney 75 CHEN et al. 2006

Gal-KO Heart, heterotopic 78* KUWAKI et al. 2005

Gal-KO Kidney 83 YAMADA et al. 2005

hCD55-KI Heart, heterotopic 99 BHATTI et al. 1999

hCD46-KI Heart, heterotopic 137 McGREGOR et al. 2005

Gal-KO Heart, heterotopic 179 TSENG et al. 2005

Gal-KO and hCD46-KI Heart, heterotopic 236 MOHIUDDIN et al. 2012

* median (MCCURRY et al. 1995) (CHEN et al. 2005) (MCGREGOR et al. 2012) (VIAL et al. 2000) (CHEN et al. 2006) (KUWAKI et al. 2005) (YAMADA et al. 2005) (BHATTI et al. 1999) (MCGREGOR et al. 2005) (TSENG et al. 2005) (MOHIUDDIN et al. 2012)

The first porcine xenografts potentially to be used for clinical applications in human are pancreatic islet cells. Porcine insulin has been successfully used for years in human patients to treat diabetes. Transplantation of porcine islets cells in chemically induced diabetic monkeys made injection of insulin dispensable. Survival of functional hCD46 porcine islet cells in monkeys lasted over 1 year (VAN DER WINDT et al. 2009). Gal-expression on porcine islet cells is age dependent; while fetal porcine islet cells express high levels of GGTA1, Gal-epitopes on adult islet cells are only occasionally detectable (BENNET et al.

2000; RAYAT et al. 2003). Using Gal-KO neonatal porcine islet (NPI) cells for xenotransplantation resulted in lower rates of primary graft dysfunction compared to wild-type (WT) NPI cells (THOMPSON et al. 2011).

After overcoming HAR by knocking out the GGTA1 gene, the major hurdle for long-term xenograft survival remains the acute vascular rejection (AVR). It was shown, that thrombotic microangiopathy is the predominant histopathological feature of rejected porcine Gal-KO cardiac xenografts (TSENG et al. 2005). This coagulation might be due to the inability of porcine tissue factor pathway inhibitor (TFPI) to adequately neutralize human factor Xa which leads to activation of endothelial cells and thrombosis (PETERSEN et al.

2009b). Transgenic pigs expressing the human TFPI are considered to effectively suppress organ rejection by its anticoagulating effect (LEE et al. 2011). The endothelial cell activation during AVR up-regulates adhesion molecules (e.g. E-selectin, VCAM-I and ICAM-I) which lead to endothelial swelling, focal ischemia and diffuse microvascular thrombosis (LEVENTHAL et al. 1993; OSBORN 1990). The human A20 (encoded by the tumor necrosis factor α-induced protein 3 gene (TNFAIP3)) expressed in transgenic pigs provides protection against tumor necrosis factor α-mediated apoptosis, and partial protection against CD95(Fas)L-mediated cell death, revealing hA20 as a promising molecule for controlling AVR in xenotransplantation studies (OROPEZA et al. 2009).

The third hurdle is the delayed xenograft rejection (DXR). This rejection is characterized by endothelial cell activation and infiltration of the graft by host monocytes and natural killer cells leading to intra-graft inflammation and thrombosis (BACH et al. 1996).

This reaction is similar to the rejection observed after allotransplantation and might be controlled by appropriate immunosuppressive agents.

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When these hurdles are overcome, there still is the risk of potential cross-species infection with the porcine endogenous retrovirus (PERV). PERVs are encoded by the porcine genome and were demonstrated to infect human cells in vitro (PATIENCE et al. 2001; SPECKE et al. 2001). However, in 160 patients, that underwent different forms of exposure to porcine tissue, no PERV infection was observed ten years after exposure (EKSER et al. 2009).

Besides, significant reduction of PERV expression was achieved by short hairpin (sh) RNA (DIECKHOFF et al. 2008; SEMAAN et al. 2012). More than 50 copies of PERV have been identified in the porcine genome and it is conceivable that the efficient targeting by zinc-finger nucleases (ZFN) could lead to a systemic PERV knockout.