Bioscience Methods 2024, Vol.15, No.4, 162-172 http://bioscipublisher.com/index.php/bm 1 63 overstated, as they pave the way for clinical trials and the eventual widespread adoption of xenotransplantation, offering hope to countless patients in need of life-saving organ transplants. 1 Background on Porcine Pathogens 1.1 Common porcine pathogens that pose risks in xenotransplantation Porcine pathogens present significant challenges in the field of xenotransplantation, where pig organs are transplanted into human recipients. Among these pathogens, porcine endogenous retroviruses (PERVs) are of particular concern. PERVs are integrated into the genome of all pigs and have the potential to infect human cells, posing a zoonotic risk (Denner, 2022). Other notable pathogens include porcine cytomegalovirus (PCMV), porcine roseolovirus (PCMV/PRV), and porcine circovirus 3 (PCV3), which have been observed to transmit during xenotransplantation procedures (Denner, 2022). 1.2 Mechanisms of pathogen transmission from pigs to humans The transmission of pathogens from pigs to humans during xenotransplantation can occur through several mechanisms. PERVs, being integrated into the pig genome, can be transmitted through the transplanted organ or tissue. These viruses are capable of infecting human cells, although no transmission has been observed in clinical trials involving pig islet cells (Denner, 2021). Other viruses, such as PCMV/PRV and PCV3, have been detected in recipients of pig heart transplants, indicating that these viruses can be transmitted through the transplanted organ (Denner, 2022). The transmission of these viruses can lead to significant complications, including reduced survival time of the xenotransplant (Denner, 2022). 1.3 Historical cases and lessons learned from previous xenotransplantation attempts Historical attempts at xenotransplantation have provided valuable insights into the risks and challenges associated with porcine pathogens. In early clinical trials, no transmission of PERVs was observed in diabetic patients receiving pig islet cells (Niu et al., 2020). However, preclinical trials involving the transplantation of pig hearts into baboons revealed the transmission of PCMV/PRV and PCV3, which significantly reduced the survival time of the xenotransplants. The first pig heart transplantation into a human patient also resulted in the transmission of PCMV/PRV, potentially contributing to the patient's death (Denner, 2021). These cases highlight the importance of rigorous screening and the development of strategies to mitigate the risk of pathogen transmission in xenotransplantation. 2 Genetic Modifications to Enhance Biosafety 2.1 Overview of genetic engineering techniques Genetic engineering has revolutionized the field of xenotransplantation, offering innovative approaches to eliminate porcine pathogens and enhance the biosafety of transplantable pig organs. Among the most prominent techniques are CRISPR/Cas9 and TALENs (Transcription Activator-Like Effector Nucleases). Techniques such as CRISPR/Cas9 have enabled precise modifications of the pig genome to reduce immunogenicity and enhance compatibility with the human immune system (Zhang, 2024). CRISPR/Cas9, a groundbreaking gene-editing tool, allows precise alterations to the pig genome by utilizing guide RNA to direct the Cas9 enzyme to specific DNA sequences, where it induces double-strand breaks that are repaired, resulting in gene disruption or modification. This technique is highly efficient, cost-effective, and versatile, making it ideal for targeting multiple genes simultaneously (Zheng et al., 2022). TALENs, on the other hand, function through engineered proteins that bind to specific DNA sequences and induce cuts, leading to gene modifications. Although TALENs are highly specific and effective for single gene edits, they are generally more labor-intensive and costly compared to CRISPR/Cas9. Both techniques have been instrumental in advancing genetic modifications aimed at reducing pathogen transmission risks in xenotransplantation (Ryczek et al., 2022). 2.2 Specific genes and pathways targeted to eliminate porcine pathogens Targeting specific genes and pathways has been crucial in the effort to eliminate porcine pathogens from transplantable organs. One of the primary targets is the porcine endogenous retroviruses (PERVs), which are integrated into the pig genome and pose significant zoonotic risks. Using CRISPR/Cas9, researchers have successfully inactivated multiple copies of PERVs, significantly reducing the potential for cross-species viral
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