International Journal of Molecular Zoology 2024, Vol.14, No.2, 72-83 http://animalscipublisher.com/index.php/ijmz 77 5 Experimental Outcomes and Clinical Trials 5.1 Summary of preclinical studies and their outcomes Preclinical studies have demonstrated significant advancements in the field of xenotransplantation using genetically modified pigs. These studies primarily focus on overcoming immune rejection and physiological incompatibilities. For instance, genetically modified pigs with deletions of specific pig genes and insertions of human complement and coagulation-regulatory transgenes have shown promising results in preventing xenograft rejection (Lei et al., 2022). Additionally, the use of porcine neonatal islet cell clusters (NICC) from genetically modified pigs has successfully normalized blood sugar levels in diabetic baboons, with graft survival exceeding 22 months (Hawthorne et al., 2022) (Figure 2). These findings underscore the potential of genetic modifications in enhancing graft survival and function in preclinical models. 5.2 Overview of current and past clinical trials involving genetically modified pigs Clinical trials involving genetically modified pigs are still in the nascent stages but have shown encouraging results. The first clinical steps have been taken to address the major barriers to xenotransplantation, such as humoral and cellular immune responses and physiological incompatibilities (Lei et al., 2022). In cardiac xenotransplantation, genetically engineered pig hearts have been transplanted into baboons, achieving significant prolongation of graft survival. For example, hearts from genetically engineered piglets expressing human complement regulatory genes and thrombomodulin have survived for over a year in some cases (Mohiuddin et al., 2014). These trials highlight the potential for genetically modified pigs to serve as viable organ donors for human transplantation in the future. 5.3 Analysis of immunotolerance and graft survival rates The analysis of immunotolerance and graft survival rates in xenotransplantation has shown that genetic modifications in donor pigs can significantly enhance graft survival. For instance, the deletion of the αGal gene and the expression of human complement regulators CD55 and CD59 in donor pigs have resulted in long-term diabetes cure in baboons, with graft survival exceeding 22 months (Hawthorne et al., 2022). Similarly, genetically engineered pig hearts with targeted immunosuppression protocols have achieved long-term survival, with some grafts surviving beyond 200 days and even surpassing the one-year mark (Mohiuddin et al., 2014). These outcomes suggest that genetic modifications, combined with appropriate immunosuppressive protocols, can effectively mitigate immune rejection and enhance graft survival in xenotransplantation. 6 Challenges and Limitations 6.1 Technical challenges in achieving stable genetic modifications Achieving stable genetic modifications in pigs for xenotransplantation presents several technical challenges. One primary issue is the complexity of introducing multiple genetic modifications that are necessary to prevent immune rejection and other complications. For instance, pigs need to be genetically engineered to lack certain xenoantigens, such as alpha-1,3-galactosyltransferase, and to express human complement and coagulation regulatory proteins like CD46 and thrombomodulin (Klymiuk et al., 2010; Mohiuddin et al., 2014; Cooper et al., 2019). Ensuring that these modifications are stably integrated and expressed at appropriate levels in the pigs' organs is technically demanding. Additionally, the combination of multiple genetic modifications can lead to unforeseen interactions and complications, making the process even more challenging (Pan et al., 2019; Carvalho-Oliveira et al., 2021). 6.2 Potential off-target effects and long-term genetic stability Another significant challenge is the potential for off-target effects and ensuring long-term genetic stability of the modifications. Techniques such as CRISPR/Cas9, while powerful, can sometimes introduce unintended mutations that may have deleterious effects (Lei et al., 2022; Xi et al., 2023). Moreover, the long-term stability of these genetic modifications is crucial for the success of xenotransplantation. There is a need for extensive in vitro and in vivo testing to ensure that the genetic modifications do not degrade over time or lead to adverse effects in the recipient (Klymiuk et al., 2010; Cooper et al., 2019). The risk of endogenous retroviruses reactivating in genetically modified pigs also poses a long-term genetic stability concern (Pan et al., 2019; Lei et al., 2022).
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