Cutting edge of genetically modified pigs targeting complement

Cutting edge of genetically

modiﬁed pigs targeting

complement activation

for xenotransplantation

Qin Sun

†

, Si-Yuan Song

†

, Jiabao Ma

†

, Danni Li

†

Yiping

Wang

, Zhengteng Yang

and Yi Wang

Department of Endocrinology, Sichuan Academy of Medical Sciences and Sichuan Provincial

People’s Hospital, School of Medicine, University of Electronic Science and Technology of China,

Chengdu, China,

Department of Neuroscience, Baylor College of Medicine, Houston, TX, United

States,

School of Pharmacy, Guangxi University of Chinese Medicine, Nanning, China,

Department

of Pharmacy, Longquanyi District of Chengdu Maternity & Child Health Care Hospital,

Chengdu, China,

Department of Critical Care Medicine, Sichuan Academy of Medical Sciences and

Sichuan Provincial People’s Hospital, School of Medicine, University of Electronic Science and

Technology of China, Chengdu, China,

Clinical Immunology Translational Medicine Key Laboratory

of Sichuan Province, Center of Organ Transplantation, Sichuan Academy of Medical Science and

Sichuan Provincial People’s Hospital, Chengdu, Sichuan, China

In the quest to address the critical shortage of donor organs for transplantation,

xenotransplantation stands out as a promising solution, offering a more abundant

supply of donor organs. Yet, its widespread clinical adoption remains hindered by

signiﬁcant challenges, chief among them being immunological rejection. Central

to this issue is the role of the complement system, an essential component of

innate immunity that frequently triggers acute and chronic rejection through

hyperacute immune responses. Such responses can rapidly lead to transplant

embolism, compromising the function of the transplanted organ and ultimately

causing graft failure. This review delves into three key areas of

xenotransplantation research. It begins by examining the mechanisms through

which xenotransplantation activates both the classical and alternative

complement pathways. It then assesses the current landscape of

xenotransplantation from donor pigs, with a particular emphasis on the

innovative strides made in genetically engineering pigs to evade complement

system activation. These modiﬁcations are critical in mitigating the discordance

between pig endogenous retroviruses and human immune molecules.

Additionally, the review discusses pharmacological interventions designed to

support tra nsplantation. By exploring the intricate relationship between the

complement system and xenotransplantation, this retrospec tive analysis not

only underscores the scientiﬁc and clinical importance of this ﬁeld but also

sheds light on the potential pathways to overcoming one of the major barriers to

the success of xenografts. As such, the insights offered here hold signiﬁcant

promise for advancing xenotransplantation from a research concept to a viable

clinical reality.

KEYWORDS

xenotransplantation, complement systems, genetically modiﬁed pigs, C3a, C3b

Frontiers in Immunology frontiersin.org01

OPEN ACCESS

EDITED BY

Lisha Mou,

Shenzhen Second People’s Hospital, China

REVIEWED BY

Jeanette Villanueva,

Victor Chang Cardiac Research Institute,

Australia

Xiaofeng Zhang,

Greenwich Hospital/Yale New Haven Health,

United States

*CORRESPONDENCE

Yi Wang

[email protected]

Zhengteng Yang

[email protected]

†

These authors have contributed equally to

this work

RECEIVED 08 February 2024

ACCEPTED 15 March 2024

PUBLISHED 04 April 2024

CITATION

Sun Q, Song S-Y, Ma J, Li D, Wang Y, Yang Z

and

Wang Y (2024) Cutting edge of

genetically modiﬁed pigs targeting

complement activation for

xenotransplantation.

Front. Immunol. 15:1383936.

doi: 10.3389/fimmu.2024.1383936

Wang. This is an open-access article distributed

under the terms of the Creative Commons

Attribution License (CC BY). The use,

distribution or reproduction in other forums

is permitted, provided the original author(s)

and the copyright owner(s) are credited and

that the original publication in this journal is

cited, in accordance with accepted academic

practice. No use, distribution or reproduction

is permitted which does not comply with

these terms.

TYPE Mini Review

PUBLISHED 04 April 2024

DOI 10.3389/fimmu.2024.1383936

1 Introduction

As of 2019, China’s organ donor registration boasted close to

1.7 million volunteers, a testament to its advancements in the ﬁeld

of organ transplantation. In that same year, China ranked as the

world’s second-largest provider of allogeneic transplants,

showcasing over 10,000 kidney and 5,000 liver transplants at the

4th China-Internation al Organ Do nation Conference (1). A

signiﬁcant policy shift in 2015 marked the transition to voluntary

organ donations from Chinese citizens as the exclusive legal source

for transplants (2), which, despite its ethical merits, has led to an

even greater deﬁcit in available human organs for transplantation

and hindered research due to the scarcity.

This backd rop has propelled xenotransplantation to the

forefront as a promising solution to this shortage. Research in

this domain has progressively moved toward identifying speciﬁc

donor species, with primates being an initial choice due to their

genetic closeness to humans. However, the use of baboon organs

has consistently resulted in patient fatalities (3), steering the

scientiﬁc focus toward pigs as suitable organ donors. Pigs, with

their comparable organ size to humans and favorable breeding

traits, are currently the focal point of xenotransplantation research

(4–6). The journey of xenotransplantation, illustrated in Figure 1,is

now directed toward the development of transgenic pigs, which are

being heralded as the next step in transplantation science.

The hyperacute rejection of transplants, primarily driven by the

complement syst em, has been a longstanding ch allenge. This

system’s activation leads to the production of active compounds

like C3a and C3b (7), which catalyze immune inﬂammation and

graft endothelial thromboembolism. The discovery of a-Gal on

graft surfaces as a trigger for complement activation has steered the

development of a-Gal knockout (a-GalKO) pigs. Chinese research

teams, such as the one led by Pandengke, have been at the helm of

creating and reﬁning a-Gal and b-Gal knockout pigs for several

generations. A milestone was achieved in June 2020 with t he

cloning of a pig possessing triple knockouts, a signiﬁcant leap

made possible by gene editing technologies targeting the

B4GalNT2 and CMAH genes (8).

The crux of this article revolves around the utilization of gene

editing to modify pig donors, aiming to mitigate the issue of

complement activation-induced hyperacute rejection post-

xenotransplantation. We discuss dual approaches to this end: the

genetic elimination of the a-Gal epitope from x enograft

endothelium and the introduction of human complement

regulatory proteins (hCRPs) into grafts via transgenesis.

Additionally, we explore the pharmaceutical avenues developed to

inhibit the complement system, a critical strategy to counter

rejection in xenotransplantation.

2 Xenograft activates the

complement system

Xenograft transplantation challenges the human immune

system, particu larly through the activation of the co mplement

system, a sophisticated network of over 50 proteins crucial for the

immune response (9). It can be activated via three primary

pathways: the classical p athway (CL), the a lternative pathway

(AP), and the lectin pathway (MBL) (10), all leading to the

potential destruction of the xenograft.

The classical pathway is initiated by the C1 complex binding to

antigen-antibody complexes, leading to the activation of C4 and C2,

and subs equently, the formation of C3 convertase (11 ). This

enzyme is pivotal in cleaving C3 into C3a and C3b, with C3b

joining with C4b2a to form C5 convertase, advancing the

complement cascade (12). In contrast, the alternative pathway,

triggered by substances like natural polysaccharides, relies on the

spontaneous hydrolysis of C3 and the formation of a ﬂuid-phase C3

FIGURE 1

Milestones in the history of xenotransplantation. This timeline graphically represents the pivotal moments and groundbreaking achievements in the

ﬁeld of xenotransplantation from the early 20th century to modern day.

Sun et al. 10.3389/fimmu.2024.1383936

Frontiers in Immunology frontiersin.org02

convertase, leading to a modest production of C3b that enhances

phagocytosis and anaphylatoxin production (13, 14). The lectin

pathway st arts with MBL binding to microorganism surface

carbohydrates, recruiting MASP-1 and MASP-2 to form C3

convertase, mirroring the classical pathway’s initial steps (15, 16).

Xenotransplantation, especially from pig donors to primate

recipients, introduces immunologi cal hurdles due to the rapid

complement-mediated response that often leads to hyperacute

rejection (HAR), characterized by graft embolism and failure (7,

17). The presence of natural antibodies in the recipient binding to

pig endothelial cell surface glycoproteins, such as a-galactosidase

(a-Gal) and N-acetylneuraminic acid hydroxylase (Neu5Gc

protein), activates the complement system, leading to clott ing,

vascular embolism, and graft failure (18, 19). Studies have shown

that pig hearts transplanted into baboons are susceptible to this

rapid rejection, with serum analysis revealing IgM-a-Gal antibodies

bound to a-Gal, triggering the complement activation pathways

(18, 19).

However, genetic engineering offers promi sing strategies to

circumvent HAR by modifying donor pigs to reduce the human

complement system’s activation effects on graft survival. Knocking

out genes encoding heterologous endothelial antigens and creating

transgenic pigs expressing hCRPs are at the fore front of these

strategies (20). In vitro studies using pancreatic islets from a-

GalKO pigs showed reduced antibody deposition and lower levels

of complement activation, suggesting a diminished role of the lectin

pathway in xenograft rejection (18, 19).

Further research into the immunological interactions between

pig tissues and primate hosts has revealed that even in the absence

of preformed natural antibodies, HAR can occur, potentially

through the alternative complement pathway (21, 22). This

indicates a complex interplay between the classical and alternative

pathways in graft rejection, where the alternative pathway may

exacerbate C3a deposition within grafts, amplifying inﬂammatory

and immune responses ( 23).

Complement proteins C3a and C5a, along with the membrane

attack complex formed via the classical and alternative pathways,

play critical roles in xenograft tissue lysis. These proteins not only

mediate inﬂammation but also activa te coagulation casca des,

contributing to the risk of thromboembolism in xenografts (24).

Studies have shown that inﬂammation induced by complement

activation can signiﬁcantly reduce the expression of porcine

thrombomodulin, an anti-in

ﬂammatory molecule, on v ascular

endothelial cells, highlighting the interconnectedness of

inﬂammation and thrombosis in xenotransplantation (25).

Addressing the challenge of HAR in xenotransplantation

requires innovative approaches to prevent complement activation.

Genetic modiﬁcations in pig donors, such as eliminating a-Gal

epitopes and introducing hCRPs, represent vital steps toward

improving graft survival and reducing complement-mediated

rejection risks. These strat egies not o nly a im to mitigat e the

immediate immunological challenges but also open new avenues

for long-term success in xenotranspl antation, poten tially

transforming it into a viable solution for organ shortages (20).

3 Genetic modiﬁcations in pigs

Pigs are optimal donors for xenotransplantation due to their

genetic, physiological, and anatomical similarities to humans,

alongside their capability for breeding in controlled environments

(26, 27). Despite these advantages, the genetic differences between

pigs and humans can lead to im munologica l discordance and

potential organ rejection. Advancements in genetic engineering

and somatic cell nuclear transfer have facilitated modiﬁcations to

the pig genome to reduce organ immunogenicity, aiming to prevent

the human immune system from rejecting pig organ transplants

(27, 28)(Figure 2). This progress is pivotal in addressing immune

rejections, with research exploring the growth of human organs

within pigs through chimeric methods, although still

predominantly in rodent models.

The risk of viral infection, particularly from porcine

endogenous retroviruses (PERVs), represents a signiﬁcant

challenge in xenotransplantation (29). Strategies to mitigate this

FIGURE 2

Process of creating gene-edited pig donors for xenotransplantation. This ﬂowchart illustrates the stages of developing gene-edited pigs for organ

donation to human recipients.

Sun et al. 10.3389/fimmu.2024.1383936

Frontiers in Immunology frontiersin.org03

risk include breeding pigs in speciﬁ c-pathogen-free (SPF)

environments and selecting pigs free from PERV-C to reduce the

risk of PERV-A/C-mediated transmission to humans (30).

Although endogenous ret roviruses r emain inactive within their

host species, causing no apparent disease, they could potentially

become active and infectious upon transmission to a recipient (27,

31, 32). Immune molecular incompatibility poses another obstacle,

with the immune system targeting foreign grafts, notably triggered

by pre-existing natural xenoantibodies recognizing Gal epitopes

(33–35). Genetically engineered pigs lacking alpha-1,3-Gal epitopes

represent a crucial step toward overcoming HAR and other forms of

immune rejection (27, 28).

Non-speciﬁc immune rea ctions, such as the instant blood-

mediated inﬂammatory reaction (IBMIR), signiﬁcantly challenge

xenogeneic islet transplantation, leading to substantial graft loss

(36). Addressing these reactions involves genetic modiﬁcations of

donor animal s, anticoagul ation t herapies, and the use of anti-

inﬂammatory treatments to preserve graft integrity and prevent

adaptive immune activation (37).

4 Genetic modiﬁcation of pigs

for xenotransplantation

The development of genetically engineered pigs marks a

signiﬁcant leap forward in addressing the challenges of

xenotransplantation from pigs to primates. Through cutting-edge

genome editing techniques, scientists have been able to introduce

precise modiﬁcations into the pig genome to mitigate xenograft

rejection and diminish the risk of interspecies infection (28).

Among the most promising modiﬁcations are the disruption of

the a-Gal and the incorporation of hCRPs, which have shown

considerable promise in preclinical studies involving pig-to-non-

human primate transplants.

Recent breakthroughs in gene editing, powered by artiﬁcial

nuclease technologies, have signiﬁcantly expanded the possibilities

for generating gene-edited pigs. These technologies, including zinc

ﬁnger nuclease (ZFN) (38), transcription activator-like eff ector

nuclease (TALEN) (39), and the CRISPR/Cas system (40–43),

have enabled not only simple gene knockouts and knock-ins but

also complex multi-gene editing, precision point mutations, and

conditional gene modiﬁcations. These advancements allow for gene

editing at various developmental stages of pigs, offering new

avenues for creating donor pigs with optimized genetic traits

for xenotransplantation.

The hCRPs play a crucial role in maintaining the delicate

balance between complement activation and inhibition. Proteins

such as decay-accelerating factor (hDAF), membrane cofactor

protein (hMCP), and reactive membrane cleavage inhibitor

(hCD59) prevent unregulated complement activity, which could

otherwise lead to continuous production of complement

components and exacerbate endothe lial damage in xeno grafts

(44). The expression of these hCRPs in donor pigs can

signiﬁcantly reduce the risk of hyperacute rejection by limiting

the formation of th e m embrane attack complex (MAC) and

mitigating complement-mediated damage.

The application of DAF (CD55), a membrane component found

on various human cells, has been explored for its potential to protect

grafts from early rejection phases (45, 46). DAF can disrupt C3

convertases on the cell sur face, effectively downreg ulating

complement activation. Studies have demonstrated that expressing

hDAF in pig islets and other tissues can enhance protection

against human complement- mediated lysis and extend graft

survival (47, 48). Similarly, the expression of human h-transferase,

an inhibitor of the alter native complement pathway, has been

shown to provide signiﬁcant protection for xenografts against

human complement atta ck, as evidenced by experiments with

transgenic pig livers transplanted into baboons (49, 50). These

genetic modiﬁcations underscore the potential of genetically

engineered pigs to overcome some of the most signiﬁcant barriers

to successful xenotransplantation.

Membrane cofactor protein (MCP, CD46) plays a crucial role in

preventing the ampliﬁcation loop of C3b deposition mediated by

alternative convertase. In an innovative approach, researchers

employed

a-GalKO pigs that were genetically modiﬁed to express

human CD46 across all tissues, including the heart, exhibiting

elevated levels of human CD46 expression. This genetic

modiﬁc ation n ot o nly prevented B cell inﬁltration but a lso

signiﬁcantly reduced T cell activity in the peripheral blood of

transplants, indicating an effective suppr ession of the T cell-

mediated response to xenoantigens (51).

Human C D59 serves as a protective mechanism against

autologous cell damage by the human complement system,

speciﬁcally by inhibiting the formation of the membrane attack

complex (MAC) during the ﬁnal stage of complement activation (7,

52). Utilizing embryonic germ (EG) cells, which unlike somatic cells

can proliferate indeﬁnitely while remaining undifferentiated, Hosup

Shim (53) developed a method to create transgenic pigs capable of

expressing human CD59. These EG cells, derived from primordial

germ cells (PGC) (54), were genetically modiﬁed with a 456 bp

fragment of the hCD59 gene, encompassing the entire cod ing

region, obtained from human ﬁbrobla st genes (55). Post-

transfection into porcine EG cells (56), these modiﬁed cells

exhibited signiﬁcantly higher mitochondrial activity when

exposed to human serum containing complement, compared to

non-transgenic controls, demonstrating enhanced survival under

HAR conditions.

The development of multi-transgenic pigs offers a promising

strategy to mitigate xenograft damage more effectively. For instance,

pig cells expressing human CD59 have shown increased resistance

to lysis by human macrophages (57). Furthermore, the expression

of a1,2-fucosyltransferase (H-transferase, HT), alongside the

knockout of the a1,3-galactosyltransferase (GT) gene, presents a

viable alternative strategy. Combining gene edits to express both

hCD59 and human HT, or to achieve a-GalKO, enhances the

protective effects against human serum, thereby improving cell and

organ survival post-transplantation (58). Transgenic pigs

expressing human CD55, CD59, and H-Transferase have shown

signiﬁcant reduction in complement-mediated graft destruction

(50), although these modiﬁcations alone could n ot compl etely

prevent humoral rejection, characterized by antibody deposition

and thrombotic microangiopathy. This suggests that while

Sun et al. 10.3389/fimmu.2024.1383936

Frontiers in Immunology frontiersin.org04

signiﬁcant strides have been made, further research is necessary to

minimize rejection mechanisms in xenotransplantation (28).

5 Complement system target drugs

for transplantation therapy

The complement system plays a crucial role in innate immunity

and immune regulation, protecting against infections and

participating in various physiological and pathological processes

(59). Despite its protective functions, dysregulated complement

activation can contribute to detrimental effects, including

inﬂammation and tissue damage. A deeper understanding of the

complement system’s components and mechanisms has spurred the

development of therapeutic drugs aimed at modulating

complement activity. These drugs target v arious complement

pathways, offering potential treatments for infectious,

inﬂammatory, traumatic, cancerous, autoimmune, or age-related

conditions, as well as preventing transplant rejection (60).

Eculizumab, the ﬁrst drug targeting the complement system,

has revolutionized the treatment landscape for diseases like

paroxysmal nocturnal hemoglobinuria (PNH), signiﬁcantly

improving patient outcomes (59, 61). In the context of organ

transplantation, the complement system is implicated in several

complications, including ischemia-reperfusion injury and antibody-

mediated rejection. Therapeutic s such as C 1-1NH (Cinryze,

Berinert, Ruconest, Cetor) and Soliris are making their way into

clinical practice, showing promise but with varying efﬁcacy levels

(62). Future research is needed to identify the most effective

complement inhibitors and devise optimal treatment strate gies.

The development programs for inhibitors targeting over a dozen

distinct complement pathways are summarized, with some already

undergoing clinical trials in both healthy volunteers and patients

(62–64). This broad spectrum of complement-targeted therapies

underscores the system’s signiﬁcance across a range of medical

conditions and its potential as a therapeutic target in transplant

medicine, where controlling complement activation could mitigate

transplant rejection and improve graft survival.

6 Conclusions and perspective

The critical shortage of human organs for transplantation is a

global challenge, and xenotransplantation has emerged as a

promising approach to address this dilemma. Genetically

engineered p igs are at the forefr ont of donor option s in

xenotransplantation, offering a viable solution to the organ

shortage crisis. Advances in gene editing technologies, such as

CRISPR/Cas9, TALEN, and somatic cell nuclear transfer (SCNT),

have signiﬁcantly propelled xenotransplantation research forward,

enabling precise genetic modiﬁcations in pig donors.

The complement system plays a dual role in xenotransplantation:

it is a key player in the immune response against porcine endothelial

cells following the binding of anti- porcine antibodies and

contributes to ischemia-reperfusion injury (IRI). Additionally, its

involvement in coagulation, inﬂammation, and the adaptive

immune response adds layers of complexity to its function in

xenograft rejection. Despite these immunobiological challenges, the

advent of genetically modiﬁed pigs, alongside an expanding array of

immunosuppressants and anti-inﬂammatory medications, is

progressively overcoming the hurdles faced by xenotransplantation.

Current genetic engineering efforts targeting compleme nt

regulatory mechanisms have effectively mitigated concerns

related to complement activation. However, there remains a

potential necessity for anti-complement and anti-inﬂammatory

interventions, especially in acute settings, to ensure the long-term

success and acceptance of xenotransplantation as a feasible solution

to the organ shortage crisis.

Author contributions

QS: Writing - original draft. QY: Writing – original draft. S-YS:

Writing – original draft. JM: Writing – original draft. DL: Writing –

original draft. YPW: Writin g – original draft. ZY: Funding

acquisition, Writing – review & editing. YW: Funding acquisition,

Writing – review & editing.

Funding

The author(s) declare that ﬁnancial support was received for the

research, authorship, and/or publication of this article. This

research was supported by the National Natural Science

Foundation of China (81802504), the Sichuan Science and

Technology Bureau (2021YFH0380 and 2023YFH0010), and a

grant from Gu angxi Science and Technology Base and Talent

Project (AD23023011).

Conﬂict of interest

The authors declare that the research was conducted in the

absence of any commercial or ﬁnancial relationships that could be

construed as a potential conﬂict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and

do not necessarily represent those of their afﬁliated organizations, or

those of the publisher, the editors and the reviewers. Any product that

may be evaluated in this article, or claim that may be made by its

manufacturer, is not guaranteed or endorsed by the publisher.

Sun et al. 10.3389/fimmu.2024.1383936

Frontiers in Immunology frontiersin.org05

References

1. National Bureau of Statistics of China. National Economy was Generally Stable in

2019 with Main Projected Targets for Development Achieved, (Beijing, China: National

Bureau of Statistics of China) (2020). Available at: https://www.stats.gov.cn/english/

PressRelease/202001/t20200117_1723398.html.

2. Li J-H, Xu X, Wang Y-F, Xie H-Y, Ch en J-Y, Dong N-G, et al. Chinese

expert consensus on organ protection of transplantation (2022 edition). Hepatobiliary &

Pancreatic Diseases International. (2022) 21(6):516–526. doi: 10.1016/j.hbpd.2022.10.010

3. Cooper DKC. A brief history of cross-species organ transplantation. Proc

(Bayl Univ Med Cent). (2012) 25(1):49–57. doi: 10.1080/08998280.2012.11928783

4. Reardon S. First pig-to-human heart transplant: what can scientists learn? Nature.

(2022) 601:305–6. doi: 10.1038/d41586-022-00111-9

5. Montgomery RA, Stern JM, Lonze BE, Tatapudi VS, Mangiola M, Wu M, et al.

Results of two cases of pig-to-human kidney xenotransplantation. N Engl J Med. (2022)

386:1889–98. doi: 10.1056/NEJMoa2120238

6. Porrett PM, Orandi BJ, Kumar V, Houp J, Anderson D, Cozette Killian A, et al.

First clinical-grade porcine kidney xenotransplant using a human decedent model. Am

J Transplant. (2022) 22:1037–53. doi: 10.1111/ajt.16930

7. Tan LA, Yu B, Sim FC, Kishore U, Sim RB. Complement activation by

phospholipids: the interplay of factor H and C1q. Protein Cell. (2010) 001:1033–49.

doi: 10.1007/s13238-010-0125-8

8. Zhang Y, Pan D, Sun X, Sun G, Wang X, Liu X, et al. Production of porcine cloned

transg enic embryos expressing green ﬂuorescent protein by somatic cell nuclear

transfer. Sci China Ser C. (2006) 49):1–8. doi: 10.1007/s11427-005-0071-5

9. Liu C-CM, Susan M, Kao AH, Navratil JS, Ahearn JM. Cell-bound complement

biomarkers for systemic lupus erythematosus: from benchtop to bedside. Rheum Dis

Clin North Am. (2010) 36:161–72. doi: 10.1016/j.rdc.2009.12.003

10. Roos A, Daha M. Antibody-mediated activation of the classical complement

pathway in xenograft rejection. Transplant Immunol. (2002) 9:257–70. doi: 10.1016/

S0966-3274(02)00042-4

11. Wallis R, Mitchell DA, Schmid R, Schwaeble WJ, Keeble AH. Paths reunited:

initiation of the classical and lectin pathwa ys of complement activ ation.

Immunobiology. (2010) 1:1–11. doi: 10.1016/j.imbio.2009.08.006

12. Ballow M. C1-Bypass complement-activation pathway in patiente with chronic

urticaria and angiosoelig. Lancet. (1975) 306:248–50. doi: 10.1016/S0140-6736(75)90963-0

13. Gtze O, Müller-Eberhard HJ. The alternative pathway of complement activation.

Adv Immunol. (1976) 24:1–35. doi: 10.1016/s0065-2776(08)60328-4

14. Merle NS, Elizabeth CS, Veronique FB, Roumenina LT. Complement system

part I a. Front Immunol. (2015) 6:1–30. doi: 10.3389/ﬁmmu.2015.00257

15. Cooper DKC, Sachs DH, Colvin RB, Shimizu A, Hisashi Y, Yamada K, et al.

Rejection of cardiac xenografts transplanted from ar,3-3omsplantedFDSLHSCRHJ

genesplantedF (GalT -alT pig s to baboons. Am J Trans plant. (2008) 8:2516–26.

doi: 10.1111/j.1600-6143.2008.02444.x

16. Kaplon RJ, Platt JL, Kwiatkowski PA, Edwards NM, Xu HE, Shah AS, et al.

Absence of hyperacute rejection in pig-to-primate orthotopic pulmonary xenografts.

Transplantation. (1995) 59:410. doi: 10.1097/00007890-199502150-00017

17. Chen RH, Kadner A, Mitchell RN, Adams DH. Mechanism of delayed rejection

in transgenic pig-to-primate cardiac xenotransplantation. J Surg Res. (2000) 90:119–25.

doi: 10.1006/jsre.2000.5864

18. Thompson P, Badell I, Lowe M, Cano J, Song M, Leopardi F, et al. Islet

xenotransplantation using gal-deﬁcient neonatal donors improves engraftment and

function. other. (2011) 11:1–20. doi: 10.1111/j.1600-6143.2011.03720.x

19. Broom C, Uknis ME. Methods of treating antibody-mediated rejection in organ

transplant patients with C1-esterase inhibitor. (Australia: Patent) (2018).

20. Butler JR, Ladowski JM, Martens GR, Tector M, Tector AJ. Recent advances in

genome editing and creation of genetically modiﬁed pigs. Int J Surgery. (2015) 23:217–22.

doi: 10.1016/j.ijsu.2015.07.684

21.SuckfüllM,MüdsamM,PieskeO,EndersG,BabicR,HammerC.

Immunohistological studies of complement activation after xenogeneic perfusion of

a working heart model. Transplant Int. (1994) 7:324–8. doi: 10.1111/j.1432-

2277.1994.tb01241.x

22. Forty J, Hasan R, Cary N, White DJ, Wallwork J. Hyperacute rejection of rabbit

hearts by human blood is mediated by the alternative pathway of complement.

Transplant Proc. (1992) 24:488.

23. Platts-Mills TAE, Ishizaka K. Activation of the alternative pathway of human

complement by rabbit cells. J Immunol . (1974) 113:348– 58. doi: 10.4049/

jimmunol.113.1.348

24. Li Y, Gong P, Kong C, Tian X. Bufalin engages in RIP1-dependent and ROS-

dependent programmed necroptosis in breast cancer cells by targeting the RIP1/RIP3/

PGAM5 pathway. Anti Cancer Drugs. (2019) 30:e0770. doi: 10.1097/

CAD.0000000000000770

25. Ochando J, Ordikhani F, Boros P, Jordan S. The innate immune response to

allotransplants: mechanisms and therapeutic potentials. Cell Mol Immunol. (2019)

16):350–6. doi: 10.1038/s41423-019-0216-2

26. Heng Z, Kaixiang X, Ninglin F, Hongye Z, Hongjiang W. Construction and

current status of gene-edited xenotransplantation pigs. Electronic J Pract Organ

Transplantation. (2018) 6:412–8.

27. Hryhorowicz M, Zeyland J, Słomski R, Lipin

ski D. Genetically modiﬁed pigs as

organ donors fo r xen ot ran sp lan ta tio n. Mol Biotechnol. (2017) 59(9-10):435–44.

doi: 10.1007/s12033-017-0024-9

28. Klymiuk N, Aigner B, Brem G, Wolf E. Genetic modiﬁcation of pigs as organ

donors for xenotransplantation. Mol Reprod Dev. (2010) 77:209–21. doi: 10.1002/

mrd.21127

29. Specke V, Rubant S, Denner J. Productive infection of human primary cells and

cell lines with porcine endogenous retroviruses. Vi rology. (2001) 2 85:177–80.

doi: 10.1006/viro.2001.0934

30. Patience C, Switzer WM, Takeuchi Y, Grifﬁths DJ, Weiss RA. Multiple groups of

novel retroviral genomes in pigs and related species. JVirol. (2001) 75:2771–5.

doi: 10.1128/JVI.75.6.2771-2775.2001

31. Denner J. Porcine Endogenous Retroviruses and Xenotransplantation. Viruses.

(2021) 13(11):2156. doi: 10.3390/v13112156

32. Denner J. Recombinant porcine endogenous retroviruses (PERVviruse a new

risk for xenotransplantation? Xenotransplantation. (2010) 17:120–0. doi: 10.1111/

j.1399-3089.2010.00573_21.x

33. Ibrahim Z, Busch J, Awwad M, Wagner R, Wells K, Cooper DKC. Selected

physiologic compatibilities and incompatibilities between human and porcine organ

systems. Xenotransplantation. (2010) 13:488– 99. doi: 10.1111/j.1399-

3089.2006.00346.x

34. Guoling L, Zhiqian X, Huaqiang Y, Zhenfang W. Research progress of transgenic

and gene-edited pigs. J South China Agric Univ . (2019) 40(5):91– 101.

35. Tanihara F, Hirata M, Otoi T. Current status of the application of gene editing in

pigs. J Reprod Dev. (2021) 67(3):177–87. doi: 10.1262/jrd.2021-025

36. Matsumoto S, Tomiya M, Sawamoto O. Current status and future of clinical islet

xenotransplantation. J Diabetes. (2016) 8(4):483–93. doi: 10.1111/1753-0407.12395

37. Zhengzhao L, Tian H, Zhiming C, Lisha M. Research progress of porcine islet

xenotransplantation. Organ transplant. (2017) 008:246–50.

38. Miller JC, Holmes MC, Wang J, Guschin DY, Lee YL, Rupniewski I, et al. An

improved zinc-ﬁnger nuclease architecture for highly speciﬁc genome editing. Nat

Biotechnol. (2007) 25:778–85. doi: 10.1038/nbt1319

39. Schmid-Burgk JL, Schmidt T, Kaiser V, Höning K, Hornung V. A ligation-

independent c loning technique for high-throughput assembly of transcription

activator-like effector genes. Nat Biotechnol

. (2013) 31(1):76–81. doi: 10.1038/nbt.2460

40. Mali P, Yang L, Esvelt KM, Aach J, Guell M, Dicarlo JE, et al. RNA-guided

human genome engineering via cas9. Science. (2013) 339 :823. doi : 10.1126/

science.1232033

41. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-cas9 for

genome engineering. (2014) 157(6):1262–78. doi: 10.1016/j.cell.2014.05.010

42. Gaj T, Gersbach CA, Barbas CF. ZFN, TALEN, and CRISPR/Cas-based methods

for genome engineering. Trends Biotechnol. (2013) 31(7):397–40 5. doi: 10.1016/

j.tibtech.2013.04.004

43. Yaoqiang H, Guoling L, Huaqiang Y, Zhenfang W. Application of gene-edited

pigs in biomedical research. Genetic. (2018) v.40:30–44.

44. Roumenina LT, Zuber J, Fre

meaux-Bacchi V. Physiological and therapeutic

complement regulators in kidney transplantation. Curr Opin Organ Transplant. (2013)

18:421–9. doi: 10.1097/MOT.0b013e32836370ce

45. Kinoshita T. Distribution of decay-accelerating factor in the peripheral blood of

normal individuals and patients with paroxysmal nocturnal hemoglobinuria. J Exp

Med. (1985) 162:75–92. doi: 10.1084/jem.162.1.75

46. Asch AS. Decay-accelerating factor is present on cultured human umbilical vein

endothelial cells. J Exp Med. (1986) 163:221–6. doi: 10.1084/jem.163.1.221

47. Yamamoto T, Iwase H, King TW, Hara H, Cooper DKC. Skin

xenotransplantation: Hist orical review and clin ical potential. Burns. (2018) 44

(7):1738–1749. doi: 10.1016/j.burns.2018.02.029

48. Schmidt P, Goto M, Mauff BL, Anegon I, Korsgren O. Adenovirus-mediated

expression of human CD55 or CD59 protects adult porcine islets from complement-

mediated cell lysis by human serum. Transplantation. (2003) 75:697–702. doi: 10.1097/

01.TP.0000053249.39753.D6

49. Young-Hee J, Chi-Hun P, Gun-Hyuk J, Yeun-Ik J, In-Sung H, Yeon-Woo J, et al.

Production of multiple transgenic yucatan miniature pigs expressing human

complement regulatory factors, human CD55, CD59, and H-transferase genes. PloS

One. (2013) 8:e63241. doi: 10.1371/journal.pone.0063241

50. Ramı

rez P, Montoya MJ, Rı

os A, Palenciano CG, Majado M, Cha

vez R, et al.

Prevention of hyperacute rejection in a model of orthotopic liver xenotransplantation

from pig to baboon using polytransgenic pig livers (CD55, CD59, and H-transferase).

Transplant Proc. (2005) 37:4103–6. doi: 10.1016/j.transproceed.2005.09.186

51. Mohiuddin MM, Corcoran PC, Singh AK, Azimzadeh A, Hoyt RF Jr., Thomas

ML, et al. B-cell depletion extends the survival of GTKO.hCD46Tgpig heart xenografts

Sun et al. 10.3389/fimmu.2024.1383936

Frontiers in Immunology frontiersin.org06

in baboons for up to 8 months. Am J Transplantation. (2012) 12:763–71. doi: 10.1111/

j.1600-6143.2011.03846.x

52. Kimberley FC, Sivasankar B, Morgan BP. Alternative roles for CD59. Mol

Immunol. (2007) 44:73–81. doi: 10.1016/j.molimm.2006.06.019

53. Ahn KS, Ji YW, Park JK, Sorrell AM, Heo SY, Kang JH, et al. Production of

human CD59-transgenic pigs by embryonic germ cell nuclear transfer. Biochem

Biophys Res Commun. (2010) 400:667–72. doi: 10.1016/j.bbrc.2010.08.125

54. Uszewski KM. Control of the complement system. Adv Immunol. (1996) 61:201–

83. doi: 10.1016/S0065-2776(08)60868-8

55. Ji YW, Ahn KS, Sorrell AM, Shin S, Shim H. Cytolytic assessment of hyperacute

rejection and production of nuclear transfer embryos using hCD46-transgenic porcine

embryonic germ cells. Zygote. (2009) 17:101–8. doi: 10.1017/S096719940800511X

56. Lee JH, Lee HJ, Nahm KM, Jeon HY, Hwang WS, Paik NW, et al. Effects of

combined expression of human complement regulatory proteins and H-transferase on

the inhibition of complement-mediated cytolysis in porcine embryonic ﬁbroblasts.

Transplant Proc. (2006) 38:1618–21. doi: 10.1016/j.transproceed.2006.02.129

57. Shim H. Isolation of pluripotent stem cells from cultured porcine primordial

germ cells. Biol Reproduction. (1997) 47:1089–95. doi: 10.1095/biolreprod57.5.1089

58. Chen CG, Salvaris EJ, Romanella M, Aminian A, Pearse MJ. Transgenic

expression of human alpha1,2-fucosyl transferase (H-transferase) prolongs mouse

heart survival in an ex vivo model of xenograft rejection. Transplantation. (1998)

65:832. doi: 10.1097/00007890-199803270-00011

59. Rother RP, R ollins SA, Mojcik C F, Brods ky RA, Bell L. Disc overy and

development of the complement inhibitor eculizumab for the treatment of

paroxysmal nocturnal hemoglobinuria. Nat Biotechnol. (2007) 25:1256. doi: 10.1038/

nbt1344

60. Melis JPM, Strumane K, Ruuls SR, Beurskens FJ, Schuurman J, Parren PWHI.

Complement in therapy and disease: Regulating the complement system with antibody-

based therapeutics. Mol Immunol. (2015) 67:117– 30. doi: 10.1016/

j.molimm.2015.01.028

61. Woodruff TM, Nandakumar KS, Tedesco F. Inhibiting the C5-C5a receptor axis.

Mol Immunol. (2011) 48:1631–42. doi: 10.1016/j.molimm.2011.04.014

62. Ricklin D, Lambris JD. New milestones ahead in complement-targeted therapy.

Semin Immunol. (2016) 28(3):208–22. doi: 10.1016/j.smim.2016.06.001

63. Morgan BP, Harris CL. Complement, a target for therapy in inﬂammatory and

degenerative diseases. Nat Rev Drug Discovery. (2015) 14(12):857–77. doi: 10.1038/

nrd4657

64. Risitano AM, Marotta S. Therapeutic complement inhibition in complement-

mediated hemolytic anemias: Past, present and future. Semin Immunol. (2016) 28

(3):223–40. doi: 10.1016/j.smim.2016.05.001

Sun et al. 10.3389/fimmu.2024.1383936

Frontiers in Immunology frontiersin.org07