International Journal of Molecular Zoology 2024, Vol.14, No.4, 244-254 http://animalscipublisher.com/index.php/ijmz 247 PERV-A/C. PERV-A and PERV-C are shown with distinct color coding: blue for PERV-A and red for PERV-C. The recombinant PERV-A/C results from a recombination event in the env gene, where segments from both PERV-A and PERV-C are combined. Additionally, the schematic illustrates the structural changes occurring in PERVs and PERV-A/C genomes after being passaged on human cells. These changes include the multimerization of repeats in the long terminal repeat (LTR) regions. The detailed depiction of the gag, pol, and env genes along with the LTR highlights the genetic complexity and adaptability of these viral genomes under different conditions. 4.2 Strategies to eliminate other pathogens (e.g., bacteria, viruses, parasites) In addition to targeting PERVs, various strategies have been developed to eliminate other pathogens from pigs used for xenotransplantation. These strategies include early weaning to prevent the transmission of porcine cytomegalovirus (PCMV) and porcine roseolovirus (PCMV/PRV), as well as the use of highly sensitive PCR-based and immunological methods for detecting and screening for numerous xenotransplantation-relevant viruses. These methods ensure that donor pigs are free from a wide range of pathogens, thereby enhancing the safety of xenotransplantation (Fishman, 2018; Denner, 2022; Lei et al., 2022). 4.3 Case studies and experimental results of pathogen elimination in pigs Several case studies and experimental results have demonstrated the effectiveness of pathogen elimination strategies in pigs. For instance, in preclinical trials involving the transplantation of pig hearts into baboons, the transmission of PCMV/PRV was observed, which significantly reduced the survival time of the xenotransplant. However, early weaning was shown to eliminate PCMV/PRV from donor pigs. Additionally, no PERV transmission was observed in clinical trials involving the transplantation of pig islet cells into diabetic humans, indicating the success of pathogen elimination strategies in these cases (Denner, 2021; Eisenson et al., 2022). 4.4 Advances in genetic engineering for pathogen-free pigs Genetic engineering has played a crucial role in creating pathogen-free pigs for xenotransplantation. By deleting genes related to the synthesis of pig-specific antigens and inserting human complement and coagulation-regulatory transgenes, researchers have been able to reduce the risk of immune rejection and physiological incompatibilities. Furthermore, the genetic modification of pigs to knock down genes related to PERVs has been a significant advancement in ensuring the safety of xenotransplantation. These genetic modifications, combined with technological breakthroughs in the biomedical field, provide a promising foundation for the future of pig-to-human xenotransplantation (Kemter et al., 2018; Niu et al., 2020). 5 Impact on Immunogenicity and Organ Compatibility 5.1 Effects of pathogen elimination on immunogenicity The elimination of pathogens, particularly porcine endogenous retroviruses (PERVs), from genetically engineered pigs has shown significant promise in reducing immunogenicity. PERVs pose a risk of cross-species transmission, which can lead to immune responses in human recipients. Recent advancements using CRISPR-Cas9 technology have enabled the production of pigs with inactivated PERVs, thereby reducing the risk of zoonotic infections and subsequent immune reactions (Figure 2) (Denner, 2022). Additionally, the elimination of other porcine viruses, such as porcine cytomegalovirus (PCMV), has been shown to improve the survival time of xenotransplants by preventing virus-induced immune responses (Yue et al., 2020). The research of Denner (2022) illustrates a process for inactivating Porcine Endogenous Retroviruses (PERVs) integrated into the pig genome using CRISPR/Cas technology. The process begins with embryonic fibroblasts containing integrated PERVs. CRISPR/Cas is applied to inactivate the PERVs by targeting and disabling the pol sequence. These modified fibroblasts are then used in somatic cell nuclear transfer (SCNT), where their nucleus is transferred into an oocyte (egg cell) that also contains the inactivated PERVs. The oocyte develops into an embryo, which is then implanted into a surrogate mother pig. The resulting piglets are born with inactivated PERVs in their genome, effectively preventing the potential transmission of these retroviruses. This method demonstrates a crucial step towards producing genetically modified pigs that are safer for xenotransplantation, reducing the risk of PERV transmission to humans.
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