CRISPR-based genetically modified scaffold-free biomaterials, including extracellular vehicles, cell sheets, cell aggregates, organoids and organs, have attracted significant attention in the fields of regenerative medicine and tissue engineering in recent years. With a wide range of applications in gene therapy, modeling disease, tissue regeneration, organ xenotransplantation, modeling organogenesis as well as gene and drug screening, they are at a critical juncture from clinical trials to therapeutic applications. Xenografts have already been tested on non-human primates and humans. However, we have to admit that a series of obstacles still need to be addressed, such as immune response, viral infection, off-target effects, difficulty in mass production, and ethical issues. Therefore, future research should pay more attention to improving their safety, accuracy of gene editing, flexibility of production, and ethical rationality. This review summarizes various types of CRISPR-based genetically modified scaffold-free biomaterials, including their preparation procedures, applications, and possible improvements.

Although CRISPR-Cas-based genome editing has made significant strides over the past decade, achieving simultaneous homozygous gene editing of multiple targets in primary cells remains a significant challenge. In this study, we optimized a coselection strategy to enhance homozygous gene editing rates in the genomes of primary porcine fetal fibroblasts (PFFs). The strategy utilizes the expression of a surrogate reporter (eGFP) to select for cells with the highest reporter expression, thereby improving editing efficiency. For simultaneous multigene editing, we targeted the most challenging site for selection, whereas other target sites did not require selection. Using this approach, we successfully obtained single-cell PFF clones (three of 10) with seven or more homozygously edited genes, including GGTA1, CMAH, B4GALNT2, CD46, CD47, THBD, and GHR Importantly, cells edited using this strategy can be efficiently used for somatic cell nuclear transfer (SCNT) to generate healthy xenotransplantation pigs in <5 months, a process that previously required years of breeding or multiple rounds of SCNT.

Genetically engineered pigs exhibit significant potential as a solution to organ scarcity in xenotransplantation. Nonetheless, a formidable challenge lies in overcoming the rejection of porcine organs by the human immune system. In this study, we generated a six-gene-edited pig model by simultaneously knocking out three major xenoantigens, GGTA1, CMAH, and β4GalNT2, along with the incorporation of coagulation regulatory factor THBD, and two complement regulatory proteins, hCD55 and hCD46, in Bama miniature pigs. These pigs exhibit genetic modifications designed to reduce xenograft rejection while maintaining normal physiology. Assessments of vital organ structure and function, including the heart, liver, spleen, lungs, and kidneys in the gene-edited pigs, showed no abnormalities. The pigs with human transgene knock-in THBD and complement genes exhibited no significant alterations in coagulation function and immune performance. These healthy features hold promise for further xenotransplantation. In conclusion, we successfully constructed six-gene-edited miniature pigs. This work serves as a valuable reference for gene editing strategies for xenotransplantation pig models.

Corneal diseases represent a significant cause of blindness worldwide, with corneal transplantation being an effective treatment to prevent vision loss. Despite substantial advances in transplantation techniques, the demand for donor corneas exceeds the available supply, particularly in developing countries. Cornea xenotransplantation has emerged as a promising strategy to address the worldwide scarcity, notably using porcine corneas. In addition to the inherent immune privilege of the cornea, the low cost of porcine breeding and the anatomical and physiological similarities between humans and pigs have made porcine corneas a viable alternative. Nonetheless, ethical concerns, specifically the risk of xenozoonotic transmission and the necessity for stringent biosafety measures, remain significant obstacles. Moreover, the success of xenotransplantation is compromised by innate and adaptive immune responses, which requires meticulous consideration and further studies. Despite these challenges, recent breakthroughs have further contributed to reducing immunogenicity while preserving the corneal architecture. Advances in genetic engineering, such as the use of CRISPR-Cas9 to eliminate critical porcine antigens, have shown promise for mitigating immune reactions. Additionally, new immunosuppressive protocols, such as have techniques like decellularization and the use of porcine-derived acellular matrices, have greatly increased graft survival in preclinical models. Future research must focus on refining immunomodulatory strategies and improving graft preparation techniques to ensure the long-term survival and safety of porcine corneal xenotransplantation in clinical trials in humans.

Inbreeding, a central theme in evolutionary and conservation biology, is a crucial practice in breeding to stabilize and enhance the specific traits or to establish inbred lines. It also carries the risk of inbreeding depression, reduced fitness, and increased potential for extinction. Nevertheless, inbreeding has been extensively studied in small and endangered populations but its effects in large domesticated animals are poorly understood. Here, we aim to investigate the genomic consequences of inbreeding in the Banna miniature inbred pig (BN), a breed that has been inbred for over 40 years.

We have sequenced 41 genomes of BN and Diannan miniature pig (DN) at high-coverage (> 31×) and combined them with published whole-genomes of swine to comprehensively investigate the genetic consequences of inbreeding. We find that BN is genetically closely related to DN, which is consistent with breeding records. All families of BN have undergone an extreme bottleneck due to intensive inbreeding, resulting in higher genomic inbreeding coefficients, reduced genetic diversity, and a lower effective population size (Ne) compare to non-inbred pigs. Furthermore, BN and DN exhibit an increased genetic load relative to Asian wild boars. Prolonged inbreeding and bottlenecks have led to some purging of deleterious mutations in BN compared to DN, and a conversion from masked load to realized load.

We present a comprehensive analysis to understand and assess the consequences of inbreeding in miniature inbred pigs from a perspective of population genomics. Utilizing genomic measurements proves effective in estimating the consequences of inbreeding, especially when a detailed and accurate historical record of pedigree are lacking. Our results provide valuable resources and a detailed perspective on the genomic impacts of inbreeding, potentially guiding efforts in breeding, breed improvement, and conservation.

During human evolution, some genes were lost or silenced from the genome of hominins. These missing genes might be the key to the evolution of humans' unique cognitive skills. An inactivation mutation in CMP-N-acetylneuraminic acid hydroxylase (CMAH) was the result of natural selection. The inactivation of CMAH protected our ancestors from some pathogens and reduced the level of N-glycolylneuraminic acid (Neu5Gc) in brain tissue. Interestingly, the low level of Neu5Gc promoted the development of brain tissue, which may have played a role in human evolution. As a xenoantigen, Neu5Gc may have been involved in brain evolution by affecting neural conduction, neuronal development, and aging.

Genetically edited pigs, modified using CRISPR-Cas9 technology, hold promise as potential sources for xenotransplantation. However, the optimal combination of genetic modifications and their expression levels for initial clinical trials remains unclear. This study investigates the generation of TKO/hCD55/hTM/hEPCR (6GE) pigs and evaluates their compatibility with human immune and coagulation systems.

The 6GE pigs were generated through iterative genome editing and F1 generation breeding. Genotyping, flow cytometry, and immunohistochemistry confirmed the knockout of GGTA1, CMAH, and B4GALNT2. Expression levels of human genes (hCD55, hTM, hEPCR) were quantified. In vitro assays using aortic endothelial cells (pAECs) from 6GE pigs assessed human serum IgM and IgG binding, complement cytotoxicity, and thrombin-antithrombin (TAT) complex levels. Blood from gene-edited pigs was used for pathophysiological analysis.

Complete knockout of GGTA1, CMAH, and B4GALNT2 was confirmed in 6GE pigs. The expression of hCD55 and hTM was approximately seven and thirteen times higher than in humans, respectively, while hEPCR levels were comparable to those in humans. In vitro, 6GE pAECs showed significantly reduced binding of human IgM and IgG compared to wild-type pAECs (IgG p<0.01, IgM p<0.0001). Similar to TKO/hCD55 pAECs, 6GE pAECs exhibited a substantial reduction in complement-mediated cytotoxicity (p<0.001) compared to TKO pAECs. Co-expression of hTM and hEPCR in 6GE pigs led to a significant decrease in thrombin-antithrombin (TAT) complex levels in co-culture with human whole blood, compared to WT (p<0.0001), TKO (p<0.01), and TKO/hCD55/hTM pigs (p<0.05). Pathophysiological analysis demonstrated excellent compatibility of 6GE pig kidneys and livers with human immune and coagulation systems. However, 6GE pigs showed increased susceptibility to infection compared to other gene-edited pigs, while TKO/hCD55 pigs were considered safe when they were all bred in a general environment.

Highly expressing hCD55, along with the co-expression of hEPCR and hTM genes, is expected to effectively reduce human complement cytotoxicity and enhance anticoagulant efficacy in genetically modified pigs. The 6GE pigs exhibited robust compatibility with human physiological and immune systems, fulfilling the criteria for clinical trials. Furthermore, it is imperative to rear donor pigs in pathogen-free (DPF) facilities to mitigate infection risks and prevent the transmission of porcine pathogens to humans.

Long noncoding RNAs (lncRNAs) are crucial in porcine preimplantation embryonic development, yet their regulatory role during zygote genome activation (ZGA) is poorly understood. We analyzed transcriptome data from porcine fetal fibroblasts (PEF), induced pluripotent stem cells (iPS), and preimplantation embryos, identifying ZGA-specific lncRNAs like XLOC-040580, and further predicted its potentially interacting genes TPRA1 and BCL2L1 via co-expression network. XLOC-040580 was knocked down by siRNA microinjection and the expression of ZGA-related genes was detected by qRT-PCR. After microinjecting siRNA targeting TPRA1 and BCL2L1 at the one-cell stage, we counted the blastocyst development rate. The blastocyst development rate was consistent with the results from si-XLOC-040580 after si-TPRA1. Through dual-luciferase reporter assays, we found that XLOC-040580 was a downstream target of TPRA1. To further elucidate the mechanism of XLOC-040580, Single-cell mRNA sequencing after XLOC-040580 knockdown revealed its regulatory network involved in embryonic developmental defects. Transcriptome analysis revealed that XLOC-040580 was specifically expressed during zygote activation. Knockdown of XLOC-040580 decreased the blastocyst development rate and reduced both the total blastocyst cell number and TE cell number. TPRA1 and BCL2L1 were specifically co-expressed with XLOC-040580 during ZGA stage, and TPRA1 could interact with the promoter region of XLOC-040580 and regulate its expression. Knockdown of TPRA1 or XLOC-040580 blocked porcine embryonic development by affecting the expression of ZGA-related genes. We found and validated that lncRNA XLOC-040580 played a key role in the ZGA process, which was regulated by TPRA1. These results implied that the functional axis of TPRA1-XLOC-040580-downstream genes involved in ZGA-related functions also coordinated early embryonic development in porcine.

The Banna miniature inbred pig (BN) is an intensively inbred line for biomedical research and xenotransplantation due to its low individual variation and stable genetic background. Although it is originated from the Diannan miniature pig (DN), substantial genetic changes have actually occurred. However, the lack of a BN reference genome has limited studies on the complete genomic architecture and utilization as a biomedical model. Here, we present a high-quality genome for BN using PacBio HiFi and Hi-C sequencing technologies, with a total length of 2.66 Gb, a scaffold N50 of 143.60 Mb, and 97.59% of the sequences anchored to chromosomes. Its BUSCO score is 96.30%, higher than porcine reference assembly and DN. The genome contains 48.49% of repeats, 19,756 protein-coding genes, and 7,207 non-coding RNAs according to our annotation. The OMArk score shows a proteome completeness and consistency of 99.58% and 93.62%, respectively. These findings indicate that the chromosome-scale genome of BN provides a valuable resource for studying the genetic basis of inbreeding, facilitating further research and clinical applications.

Chromatin organization is important for gene transcription in pig genome. However, its three-dimensional (3D) structure and dynamics are much less investigated than those in human. Here, we applied the long-read chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) method to map the whole-genome chromatin interactions mediated by CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) in porcine macrophage cells before and after polyinosinic-polycytidylic acid [Poly(I:C)] induction. Our results reveal that Poly(I:C) induction impacts the 3D genome organization in the 3D4/21 cells at the fine-scale chromatin loop level rather than at the large-scale domain level. Furthermore, our findings underscore the pivotal role of CTCF-anchored chromatin interactions in reshaping chromatin architecture during immune responses. Knockout of the CTCF-binding locus further confirms that the CTCF-anchored enhancers are associated with the activation of immune genes via long-range interactions. Notably, the ChIA-PET data also support the spatial relationship between single nucleotide polymorphisms (SNPs) and related gene transcription in 3D genome aspect. Our findings in this study provide new clues and potential targets to explore key elements related to diseases in pigs and are also likely to shed light on elucidating chromatin organization and dynamics underlying the process of mammalian infectious diseases.

The Federal Ethics Committee on Non-Human Biotechnology (ECNH) of Switzerland is an independent expert committee appointed by the Federal Council and mandated to advise the federal authorities from an ethical perspective in the field of nonhuman biotechnology and gene technology. Due to recent developments in the field of xenotransplantation after the introduction of genome editing technologies, the ECNH has commissioned an expert report on the ethical questions of xenotransplantation with a focus on animal ethics. The subject of the inquiry is, in particular, if current developments in the field of xenotransplantation raise new questions regarding ethics in the nonhuman realm or if existing questions have to be re-examined and answered anew.

An interdisciplinary approach was applied to answer this question. Based on the latest empirical results from medicine and biotechnology, xenotransplantation is analyzed and evaluated with reference to the dignity of the creature (Würde der Kreatur)-which is defined in the Swiss Federal Constitution-and the dignity of animals (Tierwürde) that is stipulated in the Swiss Animal Welfare Act and the Federal Act on Non-Human Gene Technology, as well as contemporary positions in the ethics of the human-animal relationship.

The report concludes that genome editing for xenotransplantation does not generate any qualitatively new ethical issues concerning ethics in the nonhuman realm. However, contemporary biotechnological developments must be taken as an opportunity to discuss existing ethical issues in an urgent and intensified manner, particularly regarding the significance of animals' moral standing. The lack of consideration of animal-related aspects and the neglect of current developments and the state of the art of animal ethics in the recent discussion about xenotransplantation is a scientific, ethical, and political issue because animals are most negatively affected by xenotransplantation. This is especially relevant because the contemporary state of the art in animal ethics tends to consider and protect animals more strongly than in the past.

The establishment of stable porcine embryonic stem cells (pESCs) can contribute to basic and biomedical research, including comparative developmental biology, as well as assessing the safety of stem cell-based therapies. Despite these advantages, most pESCs obtained from in vitro blastocysts require complex media and feeder layers, making routine use, genetic modification, and differentiation into specific cell types difficult. We aimed to establish pESCs with a single cell-passage ability, high proliferative potency, and stable in long-term culture from in vitro-derived blastocysts using a simplified serum-free medium.

We evaluated the establishment efficiency of pESCs from in vitro blastocysts using various basal media (DMEM/F10 (1:1), DMEM/F12, and a-MEM) and factors (FGF2, IWR-1, CHIR99021, and WH-4-023). The pluripotency and self-renewal capacity of the established pESCs were analyzed under feeder or feeder-free conditions. Ultimately, we developed a simplified culture medium (FIW) composed of FGF2, IWR-1, and WH-4-023 under serum-free conditions.

The pESC-FIW lines were capable of single-cell passaging with short cell doubling times and expressed the pluripotency markers POU5F1, SOX2, and NANOG, as well as cell surface markers SSEA1, SSEA4, and TRA-1-60. pESC-FIW showed a stable proliferation rate and normal karyotype, even after 50 passages. Transcriptome analysis revealed that pESC-FIW were similar to reported pESC maintained in complex media and showed gastrulating epiblast cell characteristics. pESC-FIW were maintained for multiple passages under feeder-free conditions on fibronectin-coated plates using mTeSR™, a commercial medium used for feeder-free culture, exhibiting characteristics similar to those observed under feeder conditions.

These results indicated that inhibition of WNT and SRC was sufficient to establish pESCs capable of single-cell passaging and feeder-free expansion under serum-free conditions. The easy maintenance of pESCs facilitates their application in gene editing technology for agriculture and biomedicine, as well as lineage commitment studies.

African swine fever (ASF) is a highly fatal viral disease that poses a significant threat to domestic pigs and wild boars globally. In our study, we aimed to explore the potential of a multiplexed CRISPR-Cas system in suppressing ASFV replication and infection. By engineering CRISPR-Cas systems to target nine specific loci within the ASFV genome, we observed a substantial reduction in viral replication in vitro. This reduction was achieved through the concerted action of both Type II and Type III RNA polymerase-guided gRNA expression. To further evaluate its anti-viral function in vivo, we developed a pig strain expressing the multiplexable CRISPR-Cas-gRNA via germline genome editing. These transgenic pigs exhibited normal health with continuous expression of the CRISPR-Cas-gRNA system, and a subset displayed latent viral replication and delayed infection. However, the CRISPR-Cas9-engineered pigs did not exhibit a survival advantage upon exposure to ASFV. To our knowledge, this study represents the first instance of a living organism engineered via germline editing to assess resistance to ASFV infection using a CRISPR-Cas system. Our findings contribute valuable insights to guide the future design of enhanced viral immunity strategies.

ASFV is currently a devastating disease with no effective vaccine or treatment available. Our study introduces a multiplexed CRISPR-Cas system targeting nine specific loci in the ASFV genome. This innovative approach successfully inhibits ASFV replication in vitro, and we have successfully engineered pig strains to express this anti-ASFV CRISPR-Cas system constitutively. Despite not observing survival advantages in these transgenic pigs upon ASFV challenges, we did note a delay in infection in some cases. To the best of our knowledge, this study constitutes the first example of a germline-edited animal with an anti-virus CRISPR-Cas system. These findings contribute to the advancement of future anti-viral strategies and the optimization of viral immunity technologies.

Significant progress has been made in kidney xenotransplantation in the past few years, and this field is accelerating towards clinical translation. Therefore, surveillance of the xenograft with appropriate tools is of great importance. Ultrasonography has been widely used in kidney allotransplantation and served as an economical and non-invasive method to monitor the allograft. However, questions remain whether the ultrasonographic criteria established for human kidney allograft could also be applied in xenotransplantation.

In the current study, we established a porcine-rhesus life sustaining kidney xenotransplantation model. The xenograft underwent intensive surveillance using gray-scale, colorful Doppler ultrasound as well as 2D shear wave elastography. The kidney growth, blood perfusion, and cortical stiffness were measured twice a day. These parameters were compared with the clinical data including urine output, chemistry, and pathological findings.

The observation continued for 16 days after transplantation. Decline of urine output and elevated serum creatinine were observed on POD9 and biopsy proven antibody-mediated rejection was seen on the same day. The xenograft underwent substantial growth, with the long axis length increased by 32% and the volume increased by threefold at the end of observation. The resistive index of the xenograft arteries elevated in response to rejection, together with impaired cortical perfusion, while the peak systolic velocity (PSV) was not compromised. The cortical stiffness also increased along with rejection.

In summary, the ultrasound findings of kidney xenograft shared similarities with those in allograft but possessed some unique features. A modified criteria needs to be established for further application of ultrasound in kidney xenotransplantation.

Genetic modification of porcine donors, combined with optimized immunosuppression, has been shown to improve outcomes of experimental xenotransplant. However, little is known about outcomes in sensitized recipients, a population that could potentially benefit the most from the clinical implementation of xenotransplantation. Here, five highly allosensitized rhesus macaques received a porcine kidney from GGTA1 (α1,3-galactosyltransferase) knockout pigs expressing the human CD55 transgene (1KO.1TG) and were maintained on an anti-CD154 monoclonal antibody (mAb)-based immunosuppressive regimen. These recipients developed de novo xenoreactive antibodies and experienced xenograft rejection with evidence of thrombotic microangiopathy and antibody-mediated rejection (AMR). In comparison, three highly allosensitized rhesus macaques receiving a kidney from GGTA1, CMAH (cytidine monophospho-N-acetylneuraminic acid hydroxylase), and b4GNT2/b4GALNT2 (β-1,4-N-acetyl-galactosaminyltransferase 2) knockout pigs expressing seven human transgenes including human CD46, CD55, CD47, THBD (thrombomodulin), PROCR (protein C receptor), TNFAIP3 (tumor necrosis factor-α-induced protein 3), and HMOX1 (heme oxygenase 1) (3KO.7TG) experienced significantly prolonged graft survival and reduced AMR, associated with dampened post-transplant humoral responses, early monocyte and neutrophil activation, and T cell repopulation. After withdrawal of all immunosuppression, recipients who received kidneys from 3KO.7TG pigs rejected the xenografts via AMR. These data suggest that allosensitized recipients may be suitable candidates for xenografts from genetically modified porcine donors and could benefit from an optimized immunosuppression regimen designed to target the post-transplant humoral response, thereby avoiding AMR.

In recent years, the xenotransplantation science has advanced tremendously, with significant strides in both preclinical and clinical research. This review intends to describe the latest cutting-edge progress in knowledge and methodologies developed to overcome potential obstacles that may preclude the translation and successful application of clinical xenotransplantation.

Preclinical studies have demonstrated that it is now possible to extend beyond two years survival of primate recipients of life saving xenografts. This has been accomplished thanks to the utilization of genetic engineering methodologies that have allowed the generation of specifically designed gene-edited pigs, a careful donor and recipient selection, and appropriate immunosuppressive strategies.In this light, the compassionate use of genetically modified pig hearts has been authorized in two human recipients and xenotransplants have also been achieved in human decedents. Although encouraging the preliminary results suggest that several challenges have yet to be fully addressed for a successful clinical translation of xenotransplantation. These challenges include immunologic, physiologic and biosafety aspects.

Recent progress has paved the way for the initial compassionate use of pig organs in humans and sets the scene for a wider application of clinical xenotransplantation.

Over the last century, there have been major landmark developments in the field of medicine, enabling us to control and cure various diseases on a larger scale. A few of these include the discovery of antibiotics, the development of vaccines, and the origin of organ and tissue transplants. The continued quest for innovation in microbiology and medicine has helped humankind save millions of lives and decrease morbidity at the global level. Genetic medicine has grown significantly in the last two decades and appears to be the next frontier of curative therapies for chronic diseases. One important landmark in genetic medicine is the development of CRISPR (clustered, regularly interspaced short palindromic repeats) technology. In this article, we describe the basic structure and function of the CRISPR-Cas9 system, which, simply put, consists of an RNA part and a protein. It works as a molecular scissor that can perform targeted cuts followed by repairs in and around the genes of interest to attain favorable translational outcomes. We focused on summarizing recent studies using CRISPR-Cas9 technology in diagnosing and treating cardiovascular disease. These studies are primarily experimental and limited to animal models. However, their results are promising enough to anticipate that this technology will undoubtedly be available in clinical medicine in the coming years. CRISPR-Cas9-mediated gene editing has been used to study and potentially treat congenital heart disease, hyperlipidemias, arrhythmogenic cardiomyopathies, and the prevention of ischemia-reperfusion injury. Despite the current progress, we recognize the several challenges this technology faces, including funding for research, improving precision and reproducible results for human subjects, and establishing protocols for ethical compliance so that it is acceptable to the scientific community and the general public.

CRISPR/Cas9 technology, combined with somatic cell nuclear transplantation (SCNT), represents the primary approach to generating gene-edited pigs. The inefficiency in acquiring gene-edited nuclear donors is attributed to low editing and delivery efficiency, both closely linked to the selection of CRISPR/Cas9 forms. However, there is currently no direct method to evaluate the efficiency of CRISPR/Cas9 editing in porcine genomes. A platform based on fluorescence reporting signals and micropattern arrays was developed in this study, to visually assess the efficiency of gene editing. The optimal specifications for culturing porcine cells, determined by the quantity and state of cells grown on micropattern arrays, were a diameter of 200 µm and a spacing of 150 µm. By visualizing the area of fluorescence loss and measuring the gray value of the micropattern arrays, it was quickly determined that the mRNA form targeting porcine cells exhibited the highest editing efficiency compared to DNA and Ribonucleoprotein (RNP) forms of CRISPR/Cas9. Subsequently, four homozygotes of the β4GalNT2 gene knockout were successfully obtained through the mRNA form, laying the groundwork for the subsequent generation of gene-edited pigs. This platform facilitates a quick, simple, and effective evaluation of gene knockout efficiency. Additionally, it holds significant potential for swiftly testing novel gene editing tools, assessing delivery methods, and tailoring evaluation platforms for various cell types.

Xenotransplantation is emerging as a vital solution to the critical shortage of organs available for transplantation, significantly propelled by advancements in genetic engineering and the development of sophisticated immunosuppressive treatments. Specifically, the transplantation of kidneys from genetically engineered pigs into human patients has made significant progress, offering a potential clinical solution to the shortage of human kidney supply. Recent trials involving the transplantation of these modified porcine kidneys into deceased human bodies have underscored the practicality of this approach, advancing the field towards potential clinical applications. However, numerous challenges remain, especially in the domains of identifying suitable donor-recipient matches and formulating effective immunosuppressive protocols crucial for transplant success. Critical to advancing xenotransplantation into clinical settings are the nuanced considerations of anesthesia and surgical practices required for these complex procedures. The precise genetic modification of porcine kidneys marks a significant leap in addressing the biological and immunological hurdles that have traditionally challenged xenotransplantation. Yet, the success of these transplants hinges on the process of meticulously matching these organs with human recipients, which demands thorough understanding of immunological compatibility, the risk of organ rejection, and the prevention of zoonotic disease transmission. In parallel, the development and optimization of immunosuppressive protocols are imperative to mitigate rejection risks while minimizing side effects, necessitating innovative approaches in both pharmacology and clinical practices. Furthermore, the post-operative care of recipients, encompassing vigilant monitoring for signs of organ rejection, infectious disease surveillance, and psychological support, is crucial for ensuring post-transplant life quality. This comprehensive care highlights the importance of a multidisciplinary approach involving transplant surgeons, anesthesiologists, immunologists, infectiologists and psychiatrists. The integration of anesthesia and surgical expertise is particularly vital, ensuring the best possible outcomes of those patients undergoing these novel transplants, through safe procedural practices. As xenotransplantation moving closer to clinical reality, establishing consensus guidelines on various aspects, including donor-recipient selection, immunosuppression, as well as surgical and anesthetic management of these transplants, is essential. Addressing these challenges through rigorous research and collective collaboration will be the key, not only to navigate the ethical, medical, and logistical complexities of introducing kidney xenotransplantation into mainstream clinical practice, but also itself marks a new era in organ transplantation.

In diploid mammals, allele-specific three-dimensional (3D) genome architecture may lead to imbalanced gene expression. Through ultradeep in situ Hi-C sequencing of three representative somatic tissues (liver, skeletal muscle, and brain) from hybrid pigs generated by reciprocal crosses of phenotypically and physiologically divergent Berkshire and Tibetan pigs, we uncover extensive chromatin reorganization between homologous chromosomes across multiple scales. Haplotype-based interrogation of multi-omic data revealed the tissue dependence of 3D chromatin conformation, suggesting that parent-of-origin-specific conformation may drive gene imprinting. We quantify the effects of genetic variations and histone modifications on allelic differences of long-range promoter-enhancer contacts, which likely contribute to the phenotypic differences between the parental pig breeds. We also observe the fine structure of somatically paired homologous chromosomes in the pig genome, which has a functional implication genome-wide. This work illustrates how allele-specific chromatin architecture facilitates concomitant shifts in allele-biased gene expression, as well as the possible consequential phenotypic changes in mammals.

Genome editing in pigs for xenotransplantation has seen significant advances in recent years. This study compared three methodologies to generate gene-edited embryos, including co-injection of sperm together with the CRISPR-Cas9 system into oocytes, named ICSI-MGE (mediated gene editing); microinjection of CRISPR-Cas9 components into oocytes followed by in vitro fertilization (IVF), and microinjection of in vivo fertilized zygotes with the CRISPR-Cas9 system. Our goal was to knock-out (KO) porcine genes involved in the biosynthesis of xenoantigens responsible for the hyperacute rejection of interspecific xenografts, namely GGTA1, CMAH, and β4GalNT2. Additionally, we attempted to KO the growth hormone receptor (GHR) gene with the aim of limiting the growth of porcine organs to a size that is physiologically suitable for human transplantation. Embryo development, pregnancy, and gene editing rates were evaluated. We found an efficient mutation of the GGTA1 gene following ICSI-MGE, comparable to the results obtained through the microinjection of oocytes followed by IVF. ICSI-MGE also showed higher rates of biallelic mutations compared to the other techniques. Five healthy piglets were born from in vivo-derived embryos, all of them exhibiting biallelic mutations in the GGTA1 gene, with three displaying mutations in the GHR gene. No mutations were observed in the CMAH and β4GalNT2 genes. In conclusion, in vitro methodologies showed high rates of gene-edited embryos. Specifically, ICSI-MGE proved to be an efficient technique for obtaining homozygous biallelic mutated embryos. Lastly, only live births were obtained from in vivo-derived embryos showing efficient multiple gene editing for GGTA1 and GHR.

An estimated 1.5 million Americans suffer from Type I diabetes mellitus, and its incidence is increasing worldwide. Islet allotransplantation offers a treatment, but the availability of deceased human donor pancreases is limited. The transplantation of islets from gene-edited pigs, if successful, would resolve this problem. Pigs are now available in which the expression of the three known xenoantigens against which humans have natural (preformed) antibodies has been deleted, and in which several human 'protective' genes have been introduced. The transplantation of neonatal pig islets has some advantages over that of adult pig islets. Transplantation into the portal vein of the recipient results in loss of many islets from the instant blood-mediated inflammatory reaction (IBMIR) and so the search for an alternative site continues. The adaptive immune response can be largely suppressed by an immunosuppressive regimen based on blockade of the CD40/CD154 T cell co-stimulation pathway, whereas conventional therapy (e.g., based on tacrolimus) is less successful. We suggest that, despite the need for effective immunosuppressive therapy, the transplantation of 'free' islets will prove more successful than that of encapsulated islets. There are data to suggest that, in the absence of rejection, the function of pig islets, though less efficient than human islets, will be sufficient to maintain normoglycemia in diabetic recipients. Pig islets transplanted into immunosuppressed nonhuman primates have maintained normoglycemia for periods extending more than two years, illustrating the potential of this novel form of therapy.

Orthotopic liver transplantation (OLT) currently serves as the sole definitive treatment for thousands of patients suffering from end-stage liver disease; and the existing supply of donor livers for OLT is drastically outpaced by the increasing demand. To alleviate this significant gap in treatment, several experimental approaches have been devised with the aim of either offering interim support to patients waiting on the transplant list or bioengineering complete livers for OLT by infusing them with fresh hepatic cells. Recently, interspecies blastocyst complementation has emerged as a promising method for generating complete organs in utero over a short timeframe. When coupled with gene editing technology, it has brought about a potentially revolutionary transformation in regenerative medicine. Blastocyst complementation harbors notable potential for generating complete human livers in large animals, which could be used for xenotransplantation in humans, addressing the scarcity of livers for OLT. Nevertheless, substantial experimental and ethical challenges still need to be overcome to produce human livers in larger domestic animals like pigs. This review compiles the current understanding of interspecies blastocyst complementation and outlines future possibilities for liver xenotransplantation in humans.

End-stage organ failure can result from various preexisting conditions and occurs in patients of all ages, and organ transplantation remains its only treatment. In recent years, extensive research has been done to explore the possibility of transplanting animal organs into humans, a process referred to as xenotransplantation. Due to their matching organ sizes and other anatomical and physiological similarities with humans, pigs are the preferred organ donor species. Organ rejection due to host immune response and possible interspecies infectious pathogen transmission have been the biggest hurdles to xenotransplantation's success. Use of genetically engineered pigs as tissue and organ donors for xenotransplantation has helped to address these hurdles. Although several preclinical trials have been conducted in nonhuman primates, some barriers still exist and demand further efforts. This review focuses on the recent advances and remaining challenges in organ and tissue xenotransplantation.

Chromatin accessibility remodeling driven by pioneer factors is critical for the development of early embryos. Current studies have illustrated several pioneer factors as being important for agricultural animals, but what are the pioneer factors and how the pioneer factors remodel the chromatin accessibility in porcine early embryos is not clear. By employing low-input DNase-seq (liDNase-seq), we profiled the landscapes of chromatin accessibility in porcine early embryos and uncovered a unique chromatin accessibility reprogramming pattern during porcine preimplantation development. Our data revealed that KLF4 played critical roles in remodeling chromatin accessibility in porcine early embryos. Knocking down of KLF4 led to the reduction of chromatin accessibility in early embryos, whereas KLF4 overexpression promoted the chromatin openness in porcine blastocysts. Furthermore, KLF4 deficiency resulted in mitochondrial dysfunction and developmental failure of porcine embryos. In addition, we found that overexpression of KLF4 in blastocysts promoted lipid droplet accumulation, whereas knockdown of KLF4 disrupted this process. Taken together, our study revealed the chromatin accessibility dynamics and identified KLF4 as a key regulator in chromatin accessibility and cellular metabolism during porcine preimplantation embryo development.

Because there is a shortage of donor kidneys, researchers are exploring the possibility of using genetically modified pig kidneys for transplantation. Approaches involving knockout of carbohydrate genes or knockin of protective proteins have been attempted to determine the best gene modifications. In this study, we utilized GalT-/-;hCD39;hCD55 and GalT-/-;hCD39;hCD46;hCD55;thrombomodulin (TBM) pigs for transplantation in nonhuman primates (NHPs). The NHPs survived for 4 weeks after kidney transplantation (4 WAT) from the GalT-/-;hCD39;hCD55 pig and for 6 WAT from the GalT-/-;hCD39;hCD46;hCD55;TBM pig. However, messenger RNA (mRNA) sequencing and immunohistochemistry analysis revealed that the 6 WAT kidney exhibited more severe apoptosis, inflammation, loss of renal function, and renal fibrosis than the 4 WAT kidney. These results indicate that additional knockin of complement regulator (hCD46) and coagulation regulator (TBM) is not enough to prevent renal damage, suggesting that improved immune suppression is needed for more prolonged survival.

Xenotransplantation holds great promise as a solution to address the critical shortage of organs, but it raises concerns regarding the potential transmission of porcine viruses to recipients, leading to infections and even zoonotic diseases. Data used in this review were mainly from literature of Pubmed database. Keywords included xenotransplantation, infection, virus, and epidemiology. The original articles and critical reviews selected were relevant to this review's theme. We review the major viral infections of concern in xenotransplantation, their risk of transmission, diagnosis, treatment, and ways to prevent infection. Then, we pivot to a comprehensive overview of the current status of xenotransplantation. In addition, we offer our own insights and recommendations for propelling xenotransplantation forward, transitioning from preclinical experiments to the critical phase of clinical trials. Viral infections pose considerable safety concerns within xenotransplantation, particularly with the possibility of emerging or currently unidentified viruses. Clinical trials serve as a crucial platform to progress the safety standards of xenotransplantation. However, further studies and dedicated efforts are required to effectively translate findings into practical applications that can improve safety measures in this field.

Pig islet xenotransplantation is a potential treatment for type 1 diabetes. We have shown that maintenance immunosuppression is required to protect genetically modified (GM) porcine islet xenografts from T cell-mediated rejection in baboons. Local expression of a depleting anti-CD2 monoclonal antibody (mAb) by the xenograft may provide an alternative solution. We have previously reported the generation of GGTA1 knock-in transgenic pigs expressing the chimeric anti-CD2 mAb diliximab under an MHC class I promoter (MHCIP). In this study, we generated GGTA1 knock-in pigs in which MHCIP was replaced by the β-cell-specific porcine insulin promoter (PIP), and compared the pattern of diliximab expression in the two lines.

A PIP-diliximab knock-in construct was prepared and validated by transfection of NIT-1 mouse insulinoma cells. The construct was knocked into GGTA1 in wild type (WT) porcine fetal fibroblasts using CRISPR, and knock-in cells were used to generate pigs by somatic cell nuclear transfer (SCNT). Expression of the transgene in MHCIP-diliximab and PIP-diliximab knock-in pigs was characterised at the mRNA and protein levels using RT-qPCR, flow cytometry, ELISA and immunohistochemistry. Islets from MHCIP-diliximab and control GGTA1 KO neonatal pigs were transplanted under the kidney capsule of streptozotocin-diabetic SCID mice.

NIT-1 cells stably transfected with the PIP-diliximab knock-in construct secreted diliximab into the culture supernatant, confirming correct expression and processing of the mAb in β cells. PIP-diliximab knock-in pigs showed a precise integration of the transgene within GGTA1. Diliximab mRNA was detected in all tissues tested (spleen, kidney, heart, liver, lung, pancreas) in MHCIP-diliximab pigs, but was not detectable in PIP-diliximab pigs. Likewise, diliximab was present in the serum of MHCIP-diliximab pigs, at a mean concentration of 1.8 μg/mL, but was not detected in PIP-diliximab pig serum. An immunohistochemical survey revealed staining for diliximab in all organs of MHCIP-diliximab pigs but not of PIP-diliximab pigs. Whole genome sequencing (WGS) of a PIP-diliximab pig identified a missense mutation in the coding region for the dixilimab light chain. This mutation was also found to be present in the fibroblast knock-in clone used to generate the PIP-diliximab pigs. Islet xenografts from neonatal MHCIP-diliximab pigs restored normoglycemia in diabetic immunodeficient mice, indicating no overt effect of the transgene on islet function, and demonstrated expression of diliximab in situ.

Diliximab was widely expressed in MHCIP-diliximab pigs, including in islets, consistent with the endogenous expression pattern of MHC class I. Further investigation is required to determine whether the level of expression in islets from the MHCIP-diliximab pigs is sufficient to prevent T cell-mediated islet xenograft rejection. The unexpected absence of diliximab expression in the islets of PIP-diliximab pigs was probably due to a mutation in the transgene arising during the generation of the knock-in cells used for SCNT.

Pig liver xenotransplantation is limited by a thrombocytopenic coagulopathy that occurs immediately following graft reperfusion. In vitro and ex vivo studies from our lab suggested that the thrombocytopenia may be the result of a species incompatibility in platelet glycosylation. Realization that platelet α-granules contain antibodies caused us to reevaluate whether the thrombocytopenia in liver xenotransplantation could occur because IgM and IgG from inside platelet α-granules bound to pig liver sinusoidal endothelial cells (LSECs). Our in vitro analysis of IgM and IgG from inside α-granules showed that platelets do carry xenoreactive antibodies that can bind to known xenoantigens. This study suggests that thrombocytopenia occurring following liver xenotransplantation could occur because of xenoreactive antibodies tethering human platelets to the pig LSEC enabling the platelet to be phagocytosed. These results suggest genetic engineering strategies aimed at reducing xenoantigens on the surface of pig LSEC will be effective in eliminating the thrombocytopenia that limits survival in liver xenotransplantation.

Despite the clinical success in vascularized composite allotransplantation (VCA), systemic immunosuppression remains necessary to prevent allograft rejection. Even with potent immunosuppressive regimens (tacrolimus, mycophenolate mofetil, and steroids), most patients experience several rejection episodes, often within the same year. The risk of systemic side effects must constantly be weighed against the risk of under-immunosuppression and, thus, acute and chronic rejection. In this context, genomic editing has emerged as a potential tool to minimize the need for toxic immunosuppressive regimens and has gained attention in the fields of solid organ transplantation and xenotransplantation. This strategy may also be relevant for the future of VCA.

We discuss the topic of genetic engineering and review recent developments in this field that justify investigating tools such as clustered regularly interspaced short palindromic repeats/Cas9 in the context of VCA.

We propose specific strategies for VCA based on the most recent gene expression data. This includes the well-known strategy of tolerance induction. Specifically, targeting the interaction between antigen-presenting cells and recipient-derived T cells by CD40 knockout may be effective. The novelty for VCA is a discovery that donor-derived T lymphocytes may play a special role in allograft rejection of facial transplants. We suggest targeting these cells prior to transplantation (e.g., by ex vivo perfusion of the transplant) by knocking out genes necessary for the long-term persistence of donor-derived immune cells in the allograft.

Despite the demonstrated feasibility of VCA in recent years, continued improvements to immunomodulatory strategies using tools like clustered regularly interspaced short palindromic repeats/Cas9 could lead to the development of approaches that mitigate the limitations associated with rejection of this life-giving procedure.

CRISPR/Cas9, a highly versatile genome-editing tool, has garnered significant attention in recent years. Despite the unique characteristics of oocytes and early embryos compared to other cell types, this technology has been increasing used in mammalian reproduction. In this comprehensive review, we elucidate the fundamental principles of CRISPR/Cas9-related methodologies and explore their wide-ranging applications in deciphering molecular intricacies during oocyte and early embryo development as well as in addressing associated diseases. However, it is imperative to acknowledge the limitations inherent to these technologies, including the potential for off-target effects, as well as the ethical concerns surrounding the manipulation of human embryos. Thus, a judicious and thoughtful approach is warranted. Regardless of these challenges, CRISPR/Cas9 technology undeniably represents a formidable tool for genome and epigenome manipulation within oocytes and early embryos. Continuous refinements in this field are poised to fortify its future prospects and applications.

The vascular endothelium of xenografted pig organs represents the initial site of rejection after exposure to recipient immune cells. In this study, we aimed to develop a promoter specific to porcine vascular endothelial cells as a step toward overcoming xenograft rejection. Transcriptome analysis was performed on porcine aortic endothelial cells (PAECs), ear skin fibroblasts isolated from GGTA knockout (GTKO) pigs, and the porcine renal epithelial cell line pk-15. RNA sequencing confirmed 243 differentially expressed genes with expression changes of more than 10-fold among the three cell types. Employing the Human Protein Atlas database as a reference, we identified 34 genes exclusive to GTKO PAECs. The endothelial cell-specific adhesion molecule (ESAM) was selected via qPCR validation and showed high endothelial cell specificity and stable expression across tissues. We selected 1.0 kb upstream sequences of the translation start site of the gene as the promoter ESAM1.0. A luciferase assay revealed that ESAM1.0 promoter transcriptional activity was significant in PAECs, leading to a 2.8-fold higher level of expression than that of the porcine intercellular adhesion molecule 2 (ICAM2) promoter, which is frequently used to target endothelial cells in transgenic pigs. Consequently, ESAM1.0 will enable the generation of genetically modified pigs with endothelium-specific target genes to reduce xenograft rejection.

Like other mammalian species, the pig genome is abundant with transposable elements (TEs). The importance of TEs for three-dimensional (3D) chromatin organization has been observed in species like human and mouse, yet current understanding about pig TEs is absent. Here, we investigated the contribution of TEs for the 3D chromatin organization in three pig tissues, focusing on spleen which is crucial for both adaptive and innate immunity. We identified dozens of TE families overrepresented with CTCF binding sites, including LTR22_SS, LTR15_SS and LTR16_SSc which are pig-specific families of endogenous retroviruses (ERVs). Interestingly, LTR22_SS elements harbor a CTCF motif and create hundreds of CTCF binding sites that are associated with adaptive immunity. We further applied Hi-C to profile the 3D chromatin structure in spleen and found that TE-derived CTCF binding sites correlate with chromatin insulation and frequently overlap TAD borders and loop anchors. Notably, one LTR22_SS-derived CTCF binding site demarcate a TAD boundary upstream of XCL1, which is a spleen-enriched chemokine gene important for lymphocyte trafficking and inflammation. Overall, this study represents a first step toward understanding the function of TEs on 3D chromatin organization regulation in pigs and expands our understanding about the functional importance of TEs in mammals.

Blood vessels that contain endothelial cells (ECs) on the surface are in direct contact with host blood and are the first target of xenograft rejection. Currently, our understanding of human anti-pig vessel immune responses is primarily based on in vitro assays using pig ECs. Therefore, it is necessary to develop an animal model that permits in vivo study of human immunological rejection of pig vessels.

Pig artery tissues (PAT) were transplanted into human immune system (HIS) mice or immunodeficient NSG mice (as controls). Intragraft human immune cell infiltration and antibody deposition were quantified using histology and immunohistochemistry. Donor antigen-specific immune responses were quantified using a mixed lymphocyte reaction and a complement-dependent killing assay.

Pig CD31+ ECs were detected and increased 2-fold from weeks 3 to 5 in PAT xenografts from immunodeficient NSG mice. However, compared with NSG mice, PAT xenografts in HIS mice had significantly lower numbers of porcine CD31+ ECs and showed a marked reduction from week 3 to week 5. PAT xenograft rejection in HIS mice is associated with intensive infiltration of human immune cells, deposition of human IgM and IgG antibodies, and the formation of a tertiary lymphoid structure. Robust donor pig antigen-specific human T cells and antibody responses were detected in PAT-transplanted HIS mice.

We have developed a humanized mouse model to evaluate human anti-pig xenoimmune responses by PAT transplantation in vivo. This model is expected to facilitate the refinement of pig gene-editing strategies (the expression on EC surface) and the testing of local immunosuppressive strategies for clinical pig organ xenotransplantation.