Clinica Chimica Acta 514 (2021) 15–23

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Clinica Chimica Acta

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Review

Kidney xenotransplantation: Recent progress in preclinical research

Xiao-Hua Yu a, c, Wen-Yi Deng a, c, Hong-Tao Jiang b, c, Tao Li b, c, Yi Wang a, b, c, *
a
Institute of Clinical Medicine, The Second Affiliated Hospital of Hainan Medical University, Haikou, Hainan 460106, China
b
Department of Organ Transplantation, The Second Affiliated Hospital of Hainan Medical University, Haikou, Hainan 460106, China
c
The Transplantation Institute of Hainan Medical University, Haikou, Hainan 460106, China

A R T I C L E I N F O A B S T R A C T

Keywords: Kidney transplantation is the most effective treatment for end-stage renal disease, but is limited by the increasing
Kidney xenotransplantation shortage of deceased and living human donor kidneys. Xenotransplantation using pig organs provides the pos­
Pigs sibility to resolve the issue of organ supply shortage and is regarded as the next great medical revolution. In the
Non-human primates
past five years, there have been sequential advances toward the prolongation of life-supporting pig kidney
Genetic modifications
xenograft survival in non-human primates, with the longest survival being 499 days. This progress is due to the
Costimulation blockade
growing availability of pigs with multi-layered genetic modifications to overcome the pathobiological barriers
and the application of a costimulation blockade-based immunosuppressive regimen. These encouraging results
bring the hope to initiate the clinical trials of pig kidney transplantation in the near future. In this review, we
summarized the latest advances regarding pig kidney xenotransplantation in preclinical models to provide a basis
for future investigation and potential clinical translation.

1. Introduction
End-stage renal disease (ESRD) is a major cause of mortality across
the world. Despite chronic hemodialysis contributes to the prolongation
of survival time, but it is limited to alleviate the symptoms. Kidney
transplantation is currently regarded as an optimal option for ESRD
treatment. However, there is a continuing worldwide shortage of
deceased and living human donor kidneys for transplantation. It is
estimated that more than 100,000 patients are waiting for kidney
transplants in the US [1]. In 2015, 16,291 kidney transplantation from
deceased donors was performed in the US, but 4761 patients died when
they were waiting for a kidney [2]. This highlights an urgent need for the
unlimited and prompt supply of transplantable kidneys.
Kidney xenotransplantation provides a feasible approach to resolve
this problem. Although non-human primates (NHPs) are closer to
humans than other animals, they are not suitable for organ-source ani­
mals because of ethical concerns, a higher risk of xenozoonosis, and
difficulties in breeding. Pig kidneys are similar to human kidneys in size,
structure and major physiological indicators, such as glomerular filtra­
tion rate and endogenous creatinine clearance rate. Pig kidneys are thus
thought to an ideal source of donor organs for clinical transplantation
[3]. Due to the evolutionary discrepancy between pigs and humans,
wild-type pig kidneys cannot be directly transplanted to humans. Sur­
vival of genetically-unmodified pig kidneys in NHPs is generally no
longer than a few minutes due to hyperacute rejection (HAR) [4]. Thus,
pigs must be genetically-modified before xenotransplantation. There are
now an estimated 40 or more genetic alterations that have been pro­
duced in pigs [5]. Moreover, pigs with up to nine genetic manipulations
are also available [6]. With significant advances in gene editing tech­
nology and immunosuppressive therapy, the outcomes of pig-to-NHP
kidney xenotransplantation have largely improved in recent years
(Fig. 1), sufficiently encouraging consideration for initial clinical trials
of pig kidney transplantation [7]. In this review, we summarized the
current knowledge about kidney xenotransplantation in preclinical

Abbreviations: ESRD, end-stage renal disease; NHPs, non-human primates; HAR, hyperacute rejection; AHXR, acute humoral xenograft rejection; Gal, galactose-
α1,3-galactose; GGTA1, α1,3-galactosyltransferase; Neu5Gc, N-glycolylneuraminc acid; CMAH, cytidine monophosphate-N-acetylneuraminic acid hydroxylase;
β4GalNT2, β-1,4N-acetylgalactosaminyl transferase 2; PBMCs, peripheral blood mononuclear cells; CRPs, complement regulation proteins; CD55, decay accelerating
factor; hCD46, membrane cofactor protein; CD59, membrane inhibitor of reactive lysis; SP-D, surfactant protein D; TF, tissue factor; CD39, ectonucleoside
triphosphate diphosphohydrolase-1; TBM, thrombomodulin; TFPI, tissue factor pathway inhibitor; EPCR, endothelial protein C receptor; HO-1, hemeoxygenase-1;
PERVs, pig endogenous retroviruses; CTLA4, cytotoxic T lymphocyte-associated protein 4; IL-7R, IL-7 receptor; ATG, anti-thymoglobulin; SLA, swine leukocyte
antigen.
* Corresponding author at: Department of Organ Transplantation, The Second Affiliated Hospital of Hainan Medical University, Haikou, Hainan 460106, China.
E-mail address: wayne0108@126.com (Y. Wang).

https://doi.org/10.1016/j.cca.2020.11.028
Received 17 April 2020; Received in revised form 26 November 2020; Accepted 30 November 2020
Available online 7 December 2020
0009-8981/© 2020 Elsevier B.V. All rights reserved.
X.-H. Yu et al. Clinica Chimica Acta 514 (2021) 15–23

research to provide an important framework for future investigation and Table 1

clinical trials. Major genetic modifications for xenotransplantation and their purpose.
Modification Purpose References
2. Genetically-modified pig for xenotransplantation GGTA1KO Deletion of Gal antigen to prevent HAR [18–21]
CMAHKO Deletion of Neu5Gc antigen to counteract AHXR [27]
The high immune incompatibility between the donor and the β4GalNT2KO Deletion of Sda antigen to counteract AHXR [28]
recipient is regarded as a major hurdle for xenotransplantation. Immu­ hCD55 Prevention of complement activation [36]
hCD46 Prevention of complement activation [37]
nological barriers comprise HAR, acute humoral xenograft rejection
hCD59 Prevention of complement activation [38]
(AHXR), cellular xenograft rejection, coagulation dysregulation and hCD47 Protection against cellular xenograft rejection [45]
inflammatory response [8]. With the improvement of gene editing hSP-D Protection against cellular xenograft rejection [46]
techniques, especially CRIPSR/Cas9, a variety of genetically-engineered hCD200 Protection against cellular xenograft rejection [47]
pigs available for xenotransplantation of cells, tissues or organs have hCD274 Protection against cellular xenograft rejection [48]
hCD39 Anticoagulation [54]
been generated to overcome these obstacles (Table 1). hTBM Anticoagulation [55–58]
hTFPI Anticoagulation [60,61]
hEPCR Anticoagulation [62–65]
2.1. Deletion of xenoreactive antigens
hHO-1 Anti-inflammation [73]
hA20 Anti-inflammation [74]
HAR belongs to humoral immune response and is defined as PERV-inactivated Inactivation of PERVs [94]
antibody-mediated complement-dependent cytotoxicity. In this process,
the binding of natural preformed anti-pig antibodies in NHPs or humans
AHXR, which develops within a few days or weeks and has similar
to xenoantigens present in pig vascular endothelial cells activates
histopathological features with HAR [24]. N-glycolylneuraminc acid
complement cascade, leading to rapid graft destruction characterized by
(Neu5Gc) and Sda have been identified as two major non-Gal antigens,
thrombosis, interstitial hemorrhage and edema [9,10]. The most
both of which are produced by cytidine monophosphate-N-
important xenoantigen causing HAR is galactose-α1,3-galactose (Gal),
acetylneuraminic acid hydroxylase (CMAH) and β-1,4N-acetylgalacto­
which is synthesized by the glycosylation enzyme α1,3-galactosyl­
saminyl transferase 2 (β4GalNT2), respectively [25,26]. New World
transferase (GGTA1) [11,12]. Gal is highly expressed in most mammals,
monkeys and Humans do not express Neu5Gc and Sda, while Neu5Gc is
including pigs and New World monkeys, but not Old World monkeys
present in Old World monkeys. Our group recently reported that there
(eg, great apes, baboons) and humans [13]. Lack of Gal results in these
was less human IgG and IgM binding to red blood cells and aortic
primates making antibodies against this foreign antigen. It is believed
endothelial cells (AECs) from GGTA1KO/CMAHKO pigs than those from
that anti-Gal antibodies are induced by Gal-expressing bacteria in the
GGTA1KO pigs [27]. Peripheral blood mononuclear cells (PBMCs) from
primates’ gastrointestinal tract during infancy [14], and account for
GGTA1/CMAH/β4GalNT2 deficient pigs displayed a significant
about 1% of circulating immunoglobulins [15].
decrease in human antibody binding compared with those from pigs
Plasmapheresis and immunoadsorption were first used to remove
lacking GGTA1 and CMAH [28]. After deletion of GGTA1, CMAH and
natural preformed anti-Gal antibodies from the potential recipient’s
β4GalNT2, the immunoreactivity of pig bioprosthetic heart valves was
plasma. However, these methods have been proven to be only partially
significantly reduced [29]. Additionally, simultaneous inactivation of
successful because the antibodies can return rapidly, thereby leading to
these three genes dramatically attenuated the binding of human anti­
AHXR [16,17]. A more effective strategy is knockout of pig GGTA1 gene
bodies to pig renal tissues [30]. These data suggest that GGTA1/CMAH/
(GGTA1KO), representing a milestone event in the xenotransplantation
β4GalNT2 triple knockout provides much greater protection against
field [18]. It has been reported that using GGTA1KO pigs as a donor,
immune rejection. However, whether this genetic manipulation can
cardiac or renal graft survival in baboons is markedly prolonged
prolong kidney graft survival is still unclear and needs to be defined in
[19–21]. GGTA1KO almost completely eliminates Gal-mediated HAR
the future studies.
and thus acts as the basis for further genetic manipulations.
Other non-Gal antigens besides Neu5Gc and Sda are involved in
In addition to Gal antigens, there are a variety of non-Gal antigens on
AHXR [31]. The microvascular system, which is composed of arterioles,
the surface of pig cells [22,23]. It has been demonstrated that non-Gal
capillaries and venules, functions as a major target during AHXR [32].
antigens can also combine with Gal-specific antibodies to trigger

Fig. 1. Maximum survival of life-supporting pig kidney grafts in NHPs (1989–2019). After transplantation of pig kidneys into NHPs, maximum survival time of life-
supporting xenografts has significantly increased from 23 days in 1989 to 499 days in 2019. There were no reports in some years.

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X.-H. Yu et al.
Recently, Zhang et al. used renal microvascular endothelial cells
(RMECs) and AECs from a GGTA1KO/CMAHKO pig to immunize cyn­
omolgus monkeys, and found that monkey serum antibody binding and
cytotoxicity to RMECs was significantly higher than to AECs [33]. These
authors further revealed that 32 pig gene products were associated with
increased binding of monkey antibodies to RMECs, suggesting that these
proteins might be potential xenoantigens when pig kidneys are trans­
planted into NHPs or humans [33]. Future studies will be required to
determine which protein plays a critical role in mediating AHXR. This
may lead to further donor modification to reinforce resistance to AHXR
and further decrease xenograft antigenicity.
2.2. Transgenic expression of human complement regulation proteins
(CRPs)
Both HAR and AHXR are largely prevented through removal of Gal
and non-Gal antigens, but activation of complement cascade still con­
tributes to immune rejection and xenograft failure [34]. Although pigs
have CRPs similar to humans, their CRPs are insufficient to protect xe­
nografts from human complement-mediated injury [35]. Thus, intro­
duction of hCRPs into organ-source pigs is required to resolve this
problem. Indeed, transgenic expression of human decay accelerating
factor (hCD55) was shown to prolong pig kidney graft survival from
days to weeks [36]. Prevention of complement activation by inserting
human membrane cofactor protein (hCD46) into pig islets successfully
blocked antibody-mediated rejection and markedly improved the
outcome of islet xenotransplantation in diabetic Cynomolgus monkeys,
maintaining normoglycemia for over 12 months [37]. Pig articular
chondrocytes expressing human membrane inhibitor of reactive lysis
(hCD59) were highly resistant to complement-mediated lysis [38].
Moreover, insertion of two or three hCRPs provided greater protection
against complement activation than transgenic expression of a single
hCRP [39,40].
Recently, Liu et al. generated GGTA1KO Diannan miniature pigs
expressing hCD55 and hCD59 using T2A-mediated polycistronic vector
systems and somatic cell nuclear transfer. They found that this triple-
gene modification was more successful in inhibiting human serum-
mediated cytolysis than GGTA1KO alone [41]. Additionally,
GGTA1KO combined with additional expression of hCD46 or hCD55
significantly reduced early kidney and heart graft failure in baboons
[42,43]. Collectively, these observations strongly suggest that
GGTA1KO pig with one or two hCRPs are a more suitable donor for
kidney and other solid organ xenotransplantation.
2.3. Transgenic expression of human regulatory factors involving innate
or adaptive immune responses
Cellular xenograft rejection usually occurs days to weeks after
transplantation and is caused by the recipient’s immune cells, including
natural killer cells, monocytes/macrophages, neutrophils, dendritic cells
and T lymphocytes [44]. At present, transgenic pigs that express human
regulatory factors of innate or adaptive immune responses are available.
For instance, transgenic expression of hCD47, a species-specific immune
inhibitory receptor on macrophages, significantly decreased the
phagocytosis of pig endothelial cells and podocytes by baboon and
human macrophages [45]. This finding suggests that inserting hCD47
into pig renal glomerular cells may be beneficial to inhibit proteinuria
after kidney xenotransplantation. Surfactant protein D (SP-D), an oli­
gometric C type lectin, plays a critical role in promoting phagocytosis by
binding to carbohydrates on microbial surfaces. Introduction of hSP-D
into pig endothelial cells was protective against THP-1 macrophage-
mediated phagocytosis [46]. Overexpression of hCD200 inhibited
macrophage phagocytic and cytotoxic activities against pig endothelial
cells [47]. Also, PBMCs from hCD274 transgenic pigs had a significantly
reduced capacity to stimulate proliferation of human CD4+ T cells [48].
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Clinica Chimica Acta 514 (2021) 15–23
2.4. Transgenic expression of human coagulation-regulatory proteins
Even after successful control of HAR, DXR and T cell response,
xenotransplantation is still confronted with thrombotic micro­
angiopathy and/or consumptive coagulopathy, which can lead to
ischemic injury, graft failure and recipient death [49]. It is well known
that coagulation dysfunction is predominately caused by (i) pig vascular
endothelial destruction due to antibody binding, complement deposition
and innate immune cells (eg, monocytes), and (ii) molecular in­
compatibilities between the pig and primate coagulation-
anticoagulation systems [50–52]. When pig vascular endothelial cells
are impaired, tissue factor (TF) is liberated into circulation and then
combines with clotting factor for thrombus formation [53].
To overcome this pathobiological barrier, a useful strategy is genetic
modification of donor animals to express human coagulation-regulatory
proteins, such as ectonucleoside triphosphate diphosphohydrolase-1
(CD39), thrombomodulin (TBM), TF pathway inhibitor (TFPI), and
endothelial protein C receptor (EPCR). CD39 is known to block throm­
bosis by degrading the platelet agonist ATP. A study by Dwyer et al.
showed that overexpression of hCD39 dramatically suppressed the
clotting of human blood [54]. TBM has a stong anticoagulant effect.
Transgenic expression of hTBM was found to decrease thrombin gen­
eration and increase coagulation time [55]. Several studies including
our own showed that additional hTBM expression prevented coagula­
tion dysregulation and consequently contributed to long-term survival
of cardiac grafts in a pig-to-baboon heterotopic heart transplantation
model [56–58]. TFPI is the most important inhibitor of TF, but there is a
molecular incompatibility between pig TFPI and human TF [59]. It was
reported that pig AECs expressing hTFPI were highly resistant to coag­
ulation triggered by antibody binding [60]. In addition, pig bone
marrow mesenchymal cells transfected with hTFPI exhibited a pro­
longed recalcification time, suggesting a potential approach to resolve
the incompatibility of pig TFPI [61].
EPCR not only has an anticoagulant effect, but also mediates anti-
inflammatory and cytoprotective signaling. Insertion of hEPCR into
pig AECs on a GGTA1KO/hCD46 background was reported to protect
against platelet aggregation [62]. Transgenic expression of hEPCR
inhibited collagen-induced thrombosis and significantly increased sur­
vival time of heterotopic cardiac xenografts in the recipients [63]. When
a baboon with a kidney graft from a GGTA1KO pig expressing hCD46,
hCD55 and hEPCR functioned for 136 days until termination due to
septic shock, there was no evidence of a consumptive coagulopathy
[64]. Another study from our group showed that additional expression
of hEPCR protected pig kidney xenografts from consumptive coagulop­
athy [65]. These findings suggest that hEPCR overexpression is suffi­
cient to avoid coagulation disregulation in kidney xenotransplantation.
2.5. Transgenic expression of human anti-inflammatory proteins
Systemic inflammatory response is another major obstacle to xeno­
transplantation [66]. Studies from our team and others have demon­
strated that a variety of pro-inflammatory cytokines and chemokines are
generated after xenotransplantation [67,68]. Moreover, pro-
inflammatory cytokines is able to facilitate coagulation activation,
aggravate immune response, and enhance resistance to immunosup­
pressive therapy [69–71]. Thus, blockade of systemic inflammation has
multiple beneficial effects on xenotransplantation.
Although anti-inflammatory drugs, such as tocilizumab (IL-6 recep­
tor blocker) and etanercept (anti-TNF-α agent), have been used to con­
trol systemic inflammatory response, their efficacy is not satisfactory in
pig-NHP-xenotransplantation models [72]. A preferable means is to
insert one or more human anti-inflammatory proteins into donor pigs.
Both hemeoxygenase-1 (HO-1) and A20 play a central role in counter­
acting inflammation. It was reported that the expression of intercellular
cell adhesion molecule-1, vascular cell adhesion molecule-1 and E-
selectin was significantly decreased in pig AECs expressing hHO-1 [73].
X.-H. Yu et al.
Similarly, hA20-transgenic pig AECs showed a significant decrease in
pro-inflammatory cytokine secretion [74]. Importantly, GGTA1KO
combined with transgenic expression of hHO-1 and hA20 protected pig
kidneys from xenograft rejection during Ex vivo perfusion with human
blood [75]. This finding suggests that GGTA1KO/hHO-1/hA20 trans­
genic pigs could function as a background for further genetic manipu­
lations toward the production of a donor pig which is clinically relevant
for kidney xenotransplantation.
3. Potential strategies to reduce cross-species transmission of
pig endogenous retroviruses (PERVs)
The transmission of pig microorganism to human recipients repre­
sents another major concern in the xenotransplantation field. Since the
organ-source pigs are bred and housed under biosecure isolation con­
ditions, the vast majority of pathogenic microorganisms can be elimi­
nated from their body. However, PERVs are one exception because they
are integrated into the genome of every pig cell, and consequently will
inevitably be transferred to the recipients with the transplanted organs,
tissues or cells [76]. Three replication-competent classes, namely PERV-
A, PERV-B and PERV-C, have been identified. Both PERV-A and PERV-B
are polytropic viruses and can infect pig and human cells, while PERV-C
is an ecotropic virus and only infects pig cells [77]. PERV-A/C, a re­
combinant form of PERV-A and PERV-C, has more potent infectivity
toward human cells due to increased receptor binding [78]. PERVs have
been shown to infect several types of human cells in vitro, such as
PBMCs, human embryonic kidney cell line HEK-293, and normal human
dermal fibroblasts [79–81]. Despite accumulating evidence has
demonstrated that no transmission of PERVs occurs during preclinical
and clinical xenotransplantation trials [82–84], a potential infection risk
should not be neglected when pig organs are transplanted to humans
[85].
Several techniques are now available to inactivate PERVs. One is
RNA interference [86,87]. Using short hairpin RNA targeting pol gene,
PERV infectivity was inhibited by 70%-80% [88]. Another is virus-
neutralizing antibodies [89,90]. The third one is microRNAs (miRNAs)
targeting various PERV genes [91]. A recent study showed that multi-
targeting miRNA against long terminal region was more successful in
inhibiting PERV replication than single-targeting miRNA [92]. Thus,
long terminal region might be a good candidate target for gene silencing
of PERVs. The fourth one is CRISPR/Cas9. In 2015, Yang et al. reported
that inactivation of pol gene using CRISPR/Cas9 led to a > 1000-fold
reduction in the transmission of PERVs to human cells [93]. Subse­
quently, PERV-inactivated pigs were successfully produced by the same
team [94]. This genetically-engineered pig can effectively avoid the
cross-species transmission of PERVs and thus serve as the basis for
further genetic manipulations to provide a safe resource of tissues and
organs for xenotransplantation.
4. Progress in immunosuppressive therapy
In addition to gene editing in donor pigs, immunosuppressive ther­
apy in the recipients is necessary for successful xenotransplantation.
Conventional immunosuppressive agents include tacrolimus, cyclo­
sporine, mycophenolate mofetil, rapamycin, and corticosteroids, all of
which are approved by the US Food and Drug Administration (FDA) for
clinical use [95]. To obtain a better efficacy, a number of novel immu­
nosuppressive drugs are currently available.
T cell activation, a complex process involving multiple cell signaling
pathways, requires primary recognition signal and an additional cos­
timulatory signal. CD28 and cytotoxic T lymphocyte-associated protein
4 (CTLA4) are prototypal costimulatory and coinhibitory cell surface
signaling molecules, respectively. They belong to the members of the
immunoglobulin superfamily and share the common ligand CD80/86.
Although blockade of CD28 costimulation by two CTLA-4 immuno­
globulin fusion proteins (abatacept and belatacept) has been clinically
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Clinica Chimica Acta 514 (2021) 15–23
approved, the overall efficacy is limited because of their collateral effect
of simultaneously blocking CTLA-4 coinhibitory signals [96]. As a
promising alternative to abatacept and belatacept, selective CD28
blockers, which directly bind to CD28 and do not impede normal coin­
hibitory signals mediated by CTLA-4, have been applied to trans­
plantation. For instance, anti-CD28 monoclonal antibody (mAb) not
only preserved CTLA-4-dependent immune regulation but also inhibited
cardiac and renal graft rejection [97]. FR104 is a novel antagonist
pegylated anti-CD28 Fab’ antibody fragment. Administration of FR104
protected against steroid-resistant acute rejection, inhibited donor-
specific antibody production, and improved graft survival in NHP kid­
ney allotransplantation model [98,99]. In addition, a humanized pegy­
lated anti-CD28 domain antibody termed lulizumab was shown to be
effective in preclinical models of allotransplantation [100,101]. More­
over, both FR104 and lulizumab were safe and well tolerated in healthy
subjects [102,103].
CD40/CD40 ligand, also known as CD154, is another important
costimulation pathway. Interaction of CD154 with CD40 leads to den­
dritic cell licensing, B cell clonal expansion, and affinity maturation
toward B cell-specific antigen [104]. Blocking the CD40/CD154 axis has
been shown to promote long-term survival of pig hearts, kidneys and
islets in NHPs [58,105,106]. However, anti-CD154 mAb has a risk of
platelet activation and resultant thromboembolism [107,108]. Recently,
a novel anti-human CD154 domain antibody (BMS-986004) lacking
crystallizable fragment binding activity has been developed to avoid
these adverse effects. In a NHP kidney transplant model, administration
of BMS-986004 effectively blocked CD40/CD154 interaction and
markedly extended allograft survival, with no evidence of thromboem­
bolism [109]. Moreove, BMS-986004 combined with conventional
immunosuppressive therapy showed a synergistic role in controlling
allograft rejection [109]. Thus, BMS-986004 may be a more promising
blocker of the CD40/CD154 costimulation pathway. There is also an
urgent need to determine whether application of BMS-986004 in xen­
otransplantation has an efficacy and safety profile similar to
allotransplantation.
Histones are basic structural components of chromatin. However,
they can be released into the circulation (known as serum histones) to
induce inflammatory response, coagulation disorder and cytotoxicity
[110,111]. Our research group reported that serum histone levels were
significantly increased when baboons undergoing pig heart, liver or
kidney transplantation developed consumptive coagulopathy or infec­
tion, and showed a positive correlation with the levels of C-reactive
protein, a marker of inflammation [112]. Given that a systemic in­
flammatory response in xenograft recipients precedes the the onset of
coagulation disturbance [70,113], serum histones may be a potential
biomarker of systemic inflammation. We also found that administration
of tocilizumab dramatically attenuated serum histone levels, and par­
thenolide (an inhibitor of nuclear factor-κB) inhibited histone-induced
platelet aggregation and endothelial cell apoptosis in vitro [112].
These results suggest that pharmacologically targeting serum histones
could be valuable for controlling inflammatory response to a pig
xenograft.
T cell depletion by antibodies is commonly used as a potent immu­
nosuppressive therapy in organ transplantation. Nevertheless, a resto­
ration of T cell homeostasis after depletion therapy causes a significant
increase in memory T cells, thereby leading to graft failure [114,115].
IL-7 signals through IL-7 receptor (IL-7R), which consists of two chains:
α chain (IL-17Rα or CD127) and γ chain. This signaling pathway plays a
central role in the proliferation of both naive and memory CD4+ and
CD8+ T cells [115,116]. Recently, Belarif et al. reported that IL-7R
blockade by an anti-IL-7Rα mAb blunted antigen-specific memory T
cell responses and alleviated skin inflammation in baboons [117].
Importantly, administration of anti-IL-7Rα mAb after T cell depletion led
to marked prolongation of skin allograft survival [118]. Thus, T cell
depletion in combination with IL-7R blockade may provide greater
protection to the transplanted tissues and organs than T cell depletion
X.-H. Yu et al.
alone.
5. Progress in pig-to-NHP kidney transplantation
The pig-to-NHP models have been used as the standard experimental
models in the xenotransplantation field. In 1989, using genetically-
unmodified (wild-type) pig kidneys, the longest survival time of life-
supporting kidney grafts was 23 days [4]. By 2004, the maximal sur­
vival of pig kidney grafts from hCD55 transgenic pigs had been extended
to 90 days in cynomolgus monkeys [119]. After GGTA1KO combined
with cotransplantation vascularized thymic tissue, pig kidney graft
survival in baboons reached 83 days in 2005 [21]. Thereafter, pig-to-
NHP kidney xenotransplantation showed only slow improvement until
2015 (Table 2).
In 2015, Higginbotham et al. transplanted kidneys from GGTA1KO/
hCD55 pigs into rhesus macaques with low or high titers of anti-pig
antibodies [120]. All recipients were subject to T cell depletion using
anti-CD4 mAb, anti-CD8 mAb combined with costimulation blockade,
either anti-CD154 mAb or belatacept. One recipient who received anti-
CD154 mAb treatment and had high titers of anti-pig antibodies only
survived 6 days, while kidney xenograft survival in two lower-titer re­
cipients treated with belatacept was extended to 14 and 21 days. Sur­
prisingly, costimulation blockade by anti-CD154 mAb led to marked
prolongation of kidney xenograft survival (>133 and >126 days) in two
low-titer animals, without evidence of rejection on biopsies sampled at
day 100. Thus, pre-transtplant antibody screening and anti-CD154 cos­
timulation blockade may be essential to long-term survival of pig kidney
grafts. At the same year, Iwase et al. transplanted a kidney from a
GGTA1KO/hCD46/hCD55/hTBM/hEPCR/hCD39 pig (both TBM and
CD39 were very poorly expressed in pig kidney) into a baboon, which
received an anti-CD40 mAb-based immunosuppressive regimen and
anti-inflammatory therapy. They found that the baboon survived 136
days with a generally stable serum creatinine until termination due to
septic shock, with no consumptive coagulopathy, protein-losing ne­
phropathy and an elicited antibody response [64]. This finding suggests
that a combination of multiple gene modifications, effective immuno­
suppressive drugs and anti-inflammatory agents is of critical importance
to prolong kidney xenograft survival.
In 2017, we achieved 237- and 260-day survival in two baboons who
underwent kidney transplantation from a GGTA1KO pig transgenic for
hCD46/hCD55/hEPCR/hTFPI/hCD47 [65]. Immunosuppressive ther­
apy was with anti-thymoglobulin (ATG) + anti-CD20 mAb (induction)
and anti-CD40 mAb + rapamycin + corticosteroids (maintenance),
which was accompanied by administration with anti-TNF-α and anti-IL-
6R mAbs to block systemic inflammation. Two animals were still healthy
with good graft function for >7–8 months, and then died of infectious
complications. There were no features of consumptive coagulopathy and
protein-losing nephropathy. In contrast, two baboons with kidneys from
a GGTA1KO/hCD46/hTBM pig lost their grafts within 12 days because
of a consumptive coagulopathy despite they received same immuno­
suppressive and anti-inflammatory therapies. These observations
Table 2
Preclinical studies of pig-to-NHP kidney xenotransplantation in the past five years.
Year Donor pig Recipient
2015 GGTA1KO/hCD46/hCD55/hTBM/hEPCR/ Baboon (n = 1)
hCD39
2015 GGTA1KO/hCD55 Rhesus macaques (n = 5)
2017 GGTA1KO/hCD46/hCD55/hEPCR/hTFPI/ Baboon (n = 2)
hCD47
GGTA1KO/hCD46/hTBM Baboon (n = 2)
2018 GGTA1KO/β4GalNT2KO Rhesus macaques (n = 6)
2019 GGTA1KO/hCD55 Rhesus macaques (n =
13)
CVF, cobra venom factor; MP, methylprednisolone; MMF, mycophenolate mofetil.
Clinica Chimica Acta 514 (2021) 15–23
suggest that transgenic expression of hEPCR and/or hCD55 in the graft is
critical importance to avoid the occurrence of coagulation
dysregulation.
In 2018, Adams et al. transplanted pig kidneys lacking GGTA1 and
β4GalNT2 into six rhesus monkeys with the least reactive cross matches
[105]. An anti-CD154 mAb-based immunosuppressive regimen was
administered to all animals. They found that three kidney xenografts
were rejected within the first week, but other three ones obtained longer
survival (35, 100 and 435 days). Pathological analysis showed that
antibody-meditated rejection remained the major cause of graft failure.
Although the reason for this difference in survival time remains to be
determined, a negative crossmatch between pigs and NHPs/human may
be an important contributor to further prolongation of kidney xenograft
survival.
There is increasing evidence that CD4+ T cells play a more important
role in mediating xenograft rejection than CD8+ T cells [121,122]. More
recently, Kim et al. used GGTA1KO/hCD55 pigs as donors and rhesus
macaques with low titers of anti-pig antibodies and selective depletion
of CD4+ or CD8+ T cells as recipients [123]. They found that kidney
xenograft survival was less than 15 days in the presence of CD8+ T cell
depletion, while CD4+ T cell depletion markedly prolonged survival
time with the longest survival reported to date in a monkey (499 days).
This indicates that CD4+ T cell depletion therapy prior to trans­
plantation may be necessary for consistent, long-term kidney xenograft
survival. In addition, their in vitro data demonstrated that the prolifer­
ation and cytotoxicity of rhesus CD4+ T cells were dependent on the
presence of swine leukocyte antigen (SLA) class II [123]. Thus, SLA class
II deletion in donor pigs may be an alternative strategy for selective
depletion of CD4+ T cells. Further in vivo studies are needed to confirm
this possibility.
6. Perspectives on the optimal genetically-modified pigs for
initial clinical kidney xenotransplantation trials
With the achievement of increasingly encouraging results in pig-to-
NHP kidney xenotransplantation models during the past few years,
increasing attention is being paid towards initiating clinical trials in the
near future. There are now a number of genetically-modified pigs
available for use as the organ-source, with some pigs expressing up to
nine genetic manipulations [6]. It is not clear, however, which combi­
nation of genetic modifications will be necessary and/or adequate for an
initial clinical pig kidney transplantation. Further, there are controver­
sial reports on the utility and efficacy of multiple transgenes in pig-to-
baboon kidney transplantation [124]. Thus, identification of optimal
combination of genetic modifications in donor pigs is critical importance
to successfully initiate a clinical kidney xenotransplantation trial.
Given that three known glycan antigens (Gal, Neu5Gc and Sda) on
pig vascular endothelial cells are regarded as major reasons leading to
humoral immune response and graft failure, GGTA1/CMAH/β4GalNT2
triple knockout should constitute the basis for any clinical trial. Ac­
cording to our own work and other studies, additional expression of
Immunosuppressive regimen Survival time (days) Reference
ATG, anti-CD20, anti-CD20, CVF, anti-CD40, 136 [64]
rapamycin, MP
Anti-CD4, anti-CD8, anti-CD154/belatacept, MMF/ 6, 21, 14,>126, [120]
steroids >133
ATG, anti-CD20, CVF, anti-CD40, rapamycin, MP 237, 260 [65]
As above 12, 12
anti-CD4, anti-CD8, anti-CD154, MMF, steroids 5, 6, 6, 35, 100, 435 [105]
anti-CD4/anti-CD8, anti-CD154, MMF, solumedrol 6 to 499 [123]
19
X.-H. Yu et al.
several protective human transgenic proteins will be important and
beneficial to further prevent rejection from developing. First, inserting
hCD46 and hCD55 helps to protect kidney grafts from complement-
mediated injury. Second, transgenic expression of hEPCR is to sup­
press thrombotic microangiopathy in the graft and/or consumptive
coagulopathy in the recipient. Third, introduction of hCD47 is to inhibit
cellular immune response. Fourth, both hHO-1 and hA20 are expressed
to avoid systemic inflammatory response. Although there are a variety of
alternative genetic modifications in donor pigs, we believe that a pig
with above nine genetic manipulations may be optimal as the source of
kidneys for a initial clinical trial of xenotransplantation.
7. Conclusion and future directions
Clinical xenotransplantation may provide an ultimate solution to
worldwide shortage of donor organs and is thus regarded as the next
great medical revolution [69,125]. With the application of gene editing
and novel immunosuppressive agents, A great advance has been made in
pig-to NHP kidney transplantation in the past five years, with life-
supporting pig kidney graft survival extending beyond 1 year. This
progress predominantly results from the increasing availability of donor
pigs with multi-layered genetic modifications and the use of novel
immunosuppressive agents, particularly costimulation blockers. It is
hoped that clinical kidney xenotransplantation trials will become a re­
ality in the near future.
Undoubtedly, there are still many questions to be resolved before
initiating such a trial. As mentioned above, a combination of nine ge­
netic manipulations should be optimal, possibly necessary, for a initial
clinical pig kidney xenotransplantation. Although the beneficial effects
of each individual manipulation is confirmed by considerable in vitro
and in vivo studies, whether such a combined manipulation affects the
health of organ-source pigs remains poorly understood and requires
further characterization. Although PERV-inactivated pigs have been
produced, the efficacy and safety of PERV inactivation need to be
evaluated in pig-to-NHP xenotransplantation models. Given that cos­
timulation blockade agents are not approved by FDA for clinical use, it is
important to explore whether kidney xenograft rejection can be effec­
tively inhibited by FDA-approved pharmacologic or biologic agents.
Current clinical laboratory tests involving transplantation include as
much as human leukocyte antigen (HLA) typing, donor-specific HLA
antibody and MICA testing, renal allograft pathology, blood test for
cytomegalovirus, and urinalysis for BK virus infections. Improving the
quality of these laboratory tests and developing novel assays is essential
for clinical application of kidney xenotransplantation. During the past
decades, dual kidney transplantation has enabled greater use of mar­
ginal kidneys and reduced waiting time. Although there are no reports
on dual kidney transplantation in pig-to-NHP kidney xeno­
transplantation to date, future preclinical research should also explore
the feasibility of this transplantation. Answers to these questions will
provide insightful knowledge about xenotransplantation and contribute
to the success of initial clinical pig kidney transplantation trials.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This work was supported by grant from the major science and
technology project of Hainan Province, China (ZDKJ2019009).
20
Clinica Chimica Acta 514 (2021) 15–23
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