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Clinical Infection & Immunity

Review

Volume 7, Number 1, March 2022, pages 4-9

The Miniature Swine in Organ Transplantation: Promises and Impasses

Cross-species transplantation, called xenotransplantation, offers the prospect of an unlimited supply of organs for clinical transplantation, as an attempt to deal with the increasing demand and the shortage of human organ donors [1]. It is estimated that roughly 20 Americans die each day waiting to receive an organ [2]. Although many initial transplants for various organs used nonhuman primates as a source in the 20th century, the pig presents a series of advantages over primates, especially in terms of supply [1].

Besides their extensive use in agriculture and their more recent trend as companion animals, porcine species are now being used for biomedical research [3]. The miniature pig shares many anatomical and physiological similarities with humans [3], making it a potential source of suitable organs for xenotransplantation [4] (Fig. 1). Xenograft skin for wound coverage using pig skin as a temporary covering until autograft was accessible became popular because of the limited availability and high expense of human skin tissue.

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Figure 1. With an increase in demand and shortage for human organs, research is underway to genetically engineer pigs to evade human immune system and avoid infections in humans. Corneal cells, lungs, heart, liver, pancreas, kidneys and skins grafts are being considered for xenotransplantation.

There are several strains of minipigs [5]. Although most investigations have used young pigs (less than 1-year-old) [6], besides body size-matching, age-appropriateness is important for the efficacy of transplantation [7].

Owing to the development of gene-editing technologies, the generation of genetically modified pigs has dramatically expanded [8]. Somatic cell nuclear transfer (SCNT) using genetically modified somatic cells was the primary method for the generation of genetically modified pigs (Figs. 2b, 3b). This is a laborious and time-consuming process of limited efficiency. Recent improvements in gene-editing systems, such as the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system allow for the efficient introduction of specific modifications into cells via gene editors, reducing the effort and time required to generate genetically modified pigs [8]. In addition, direct modification of genomic DNA in zygotes or embryos using cytoplasmic microinjection and electroporation has been made possible (Fig. 2a, c). With the advent of genetic engineering and cloning technologies, pigs are available with several different manipulations that protect their tissues from the human immune response, increasing porcine graft survival in nonhuman primate models (Fig. 3).

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Figure 2. Three major methods used to generate genetically modified pigs. (a) Cytoplasmic microinjection of CRISPR/Cas modified construct into porcine zygotes. (b) Somatic cell nuclear transfer (SCNT). (c) Introduction of gene editors via electroporation. CRISPR: clustered regularly interspaced short palindromic repeats.

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Figure 3. Process of editing using the CRISPR/Cas system for xenotransplantation using two methods. (a) Cytoplasmic microinjection of CRISPR/Cas modified construct into porcine zygotes. (b) Depiction of somatic cell nuclear transfer (SCNT). CRISPR: clustered regularly interspaced short palindromic repeats.

There are several immunological and pathophysiological problems associated with pig xenotransplantation. Xenoantigens can cause a type of humoral rejection known as hyperacute rejection, within minutes to hours mediated by naturally pre-existing antibodies in the recipient against xenograft epitopes on porcine endothelial cells triggering the destruction of the graft vasculature and subsequent graft failure via the complement proteins activation [9, 10]. This has led to the efforts in removing xenoantigen biosynthetic genes [8, 11-16] using SCNT and gene-editing technology.

Innate and adaptive immune responses mediate cellular xenograft rejection via natural killer (NK) cells [17, 18], macrophages [19, 20], neutrophils, dendritic cells, T cells, and B cells, which may occur days to weeks after transplantation [21]. Xenoantibodies bind to donor cells with their Fab portion. Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) is a molecule that blocks the B7-cluster of differentiation (CD)28 costimulatory pathway in T cell activation [22]. The transgenic pig with neuronal expression of an hCTLA4-Ig gene demonstrated that hCTLA4-Ig protein reduced the proliferation of human T cells against porcine cells [23]. The benefits of the hCTL4-Ig expression were demonstrated by extended xenograft survival time in a rat skin transplantation model [24], and in a nonhuman primate neuronal transplantation model [25]. T-cell response can also be alleviated by deleting the swine leukocyte antigen (SLA) class I [26], or by introducing a mutant human class II transactivator gene (CIITA-DN) [27] in pigs. Though not primarily intended, lacking α-Gal antigens or expression of hCRPs has been demonstrated to reduce T-cell response to pig cells [28, 29]. Further in vivo evaluation is required to better understand the roles of cellular xenograft rejection [9], and modified genes in protecting xenograft from rejection response.

Sequestration of human platelets causing lethal thrombocytopenia accompanied by porcine liver xenotransplantation is another major barrier caused by binding of platelets to the asialoglycoprotein receptor (ASGR) on porcine sinusoidal endothelial cells and phagocytosis [30]. Targeted gene disruption of the ASGR-1 gene using transcription activator-like effector nucleases in pigs was shown to decrease the human platelet uptake and may prevent xenotransplantation-induced thrombocytopenia [31].

Thanks to these gene-editing technologies, various pigs are currently available with a number of different manipulations that protect their tissues from the human immune response, resulting in increasing porcine graft survival in nonhuman primate models. This has brought us closer to bridging cross-species molecular incompatibilities, and the pace of current progress may soon make the widespread clinical application of xenotransplantation become a reality.

There is some concern that a porcine microorganism might be transferred along with the transplanted organ. Porcine hepatitis E virus transmission to humans is possible [32]. Of particular attention, porcine endogenous retrovirus (PERV) genomes can be transmitted to humans [33, 34]. All swine have PERV proviruses in their genomes, and some pigs produce exogenously infectious PERV. Zoonotic PERV infection may contribute to a variety of disorders including cancer, and immunological and neurological disorders [35]. Several reports suggest that the risk of PERV infections in human recipients is less likely [36]. A 5-year monitoring for PERV infection of eight patients who were treated with a porcine cell-based bioartificial liver, was unable to detect circulation virus DNA or RNA [37]. Same thing happened in long-term PERV monitoring following islet cell transplantation in patients, where no evidence of PERV transmission was found [38, 39]. A possible explanation might be that even the PERVs cannot infect certain cell types because of the absence of a functional receptor on most cell surfaces [40]. However, in vitro PERV transmission of pig-to-human and human-to-human cells was detected [41, 42].

Ideally, donor animals would be free of all known pathogenic organisms than the average deceased human donor. Generation of PERV knockout swine using CRISPR-Cas9 technology could completely exclude PERV transmission [36]. Nevertheless, a novel unknown microorganism may still be transferred from the donor to the recipient with the graft.

Pancreatic islet transplantation is a promising treatment for type 1 diabetic patients. Although porcine islets can be produced in sufficient quantities [43], and pig islet function has been achieved when xenotransplanted to nonhuman primates, long-term xenograft function (beyond 6 months) has not been reported [44]. Attempts for pig kidney transplant have been even less encouraging [45, 46].

Using transgene expression in pig tissues, treatments of skin defects using cell therapy-based approaches that take advantage of similarities between pig and human epidermis have been achieved, and neurotransplantation using porcine neural stem cells grafted into inbred miniature pigs as an alternative model to nonhuman primates xenografted with human cells [45, 47].

The variability of gene expression in equivalent human and minipig tissues is considerably higher in minipig organs, which is important for study design in case a human target belongs to this variable category in the minipigs [48]. The first successful heart transplanted to a human from a genetically modified pig has been the subject of recent scientific and medical news [49]. This milestone in organ transplantation offers hope for thousands in need of organs.

Genetically modified pigs will inevitably be used as donor animals for organ transplantation in humans. Combining gene-editing and immunosuppressive therapy is necessary for successful xenotransplantation of different organs. In the future, optimal genetically engineered pigs and targeted immunosuppressive regimen strategy will collectively solve the problem of human organ shortage.

Acknowledgments

None to declare.

Financial Disclosure

None to declare.

Conflict of Interest

None to declare.

Author Contributions

JC and AK wrote the article.

Data Availability

Any inquiries regarding supporting data availability of this study should be directed to the corresponding author.

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