In a recent study published in Nature, a team of researchers from the United States used a genetically engineered porcine donor to develop kidney grafts, which were then transplanted into a non-human primate model to test its life-supporting function in the long term.
Study: Design and testing of a humanized porcine donor for xenotransplantation. Image Credit: David Tadevosian/Shutterstock.com
Background
Xenotransplantation, using non-human tissues, organs, or cells for medical applications on humans, is a promising answer to the organ shortage worldwide.
However, for xenografts to be ready for clinical use, they must be tested in robust clinical trials. This can occur only after xenografts developed in genetically engineered porcine donors are tested for efficacy and safety in non-human primate models.
While kidney tissues engineered in porcine donors have been tested in Old-World monkeys, and these studies have contributed significantly to understanding the challenges in xenotransplantation, these organs are not yet ready for clinical use.
Some of the challenges faced thus far include incompatibility in the size of the xenograft organ since they have been developed in commercial breeds of pigs and are too large to replace human organs.
Furthermore, the differences in the number of glycans in non-human primates and humans also result in incompatibility issues, and often, these grafts do not express all the human transgenes. The presence of endogenous retroviral sequences (PERV) in the porcine donors also presents a risk of zoonotic disease.
About the study
In the present study, the researchers used a breed of humanized Yucatan miniature pigs as the porcine donor to develop renal grafts without the three major glycans and with the porcine retroviral genes inactivated.
The grafts were also engineered to overexpress human transgenes. These renal grafts were transplanted into Macaca fascicularis or cynomolgus monkeys, which served as the non-human primate models to test the efficacy and safety of the renal grafts.
The cell surface of porcine cells presents three major glycans produced by three genes involved in glycan synthesis — cytidine monophospho-Nacetylneuraminic acid hydroxylase, glycoprotein α-galactosyltransferase 1, and β-1,4-N-acetylgalactosaminyltransferase 2.
Humans and other primates, such as Old-World monkeys, have preformed antibodies against these three glycan antigens, which result in antibody-mediated rejection of xenografts expressing the three glycans.
A Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) single guide ribonucleic acid (RNA) guide design was used to target the genes coding for the three main glycans expressed on porcine cell surfaces.
The nucleus from cells that were edited were subsequently used for somatic cell nuclear transfer (SCNT) to produce pigs that had these edits. The pigs made after SCNT were all female Yucatan miniature pigs (Sus scrofa domesticus).
Immunohistochemical staining was used to compare eight-week-old kidney samples from wild-type and transgenic Yucatan pigs to determine the expression of human proteins such as thrombomodulin, CD46, CD47, CD55, endothelial protein C receptor, etc.
Additionally, the mean fluorescent intensities were measured for the transgene proteins from the glomeruli, blood vessels, tubules, and whole kidney tissues.
Various assays and measurements such as glomerular filtration analysis, cytotoxicity assay, thrombin-antithrombin III (TAT) complex assay, analysis of primate anti-porcine immunoglobulin (Ig) M and IgG, and many more were conducted to assess the compatibility and safety of the xenografts.
The kidney xenografts were transplanted into male and female M. fascicularis cynomolgus monkeys and postoperatively monitored through ultrasound, urine output, hematology, clinical chemistry, and histopathological analyses of renal biopsies.
Results
The results reported that the transgenic porcine donor carried 69 genomic edits, including eliminating the three major surface glycans, inactivating porcine endogenous retroviral genes, and overexpressing human transgenes.
The various functional analyses conducted in vitro suggested the modulation of inflammation by the genetically edited renal endothelial cells was strong enough to make them difficult to distinguish from human endothelial cells, indicating a high level of compatibility with the human immune system.
The kidneys that had only the three porcine glycan genes knocked out demonstrated poor graft survival after being transplanted into the non-human primate model. In comparison, those edited to knock out the three glycan genes and overexpress human transgenes exhibited significantly better survival in the cynomolgus monkeys.
The researchers reported that although the knocking out of the three major glycan-producing genes significantly decreased the preformed antibody binding to the porcine antigens, residual antibody binding was still observed due to the remaining minor xenoantigens.
Furthermore, the cytotoxicity assay reported higher levels of cytotoxic activity in sera from the Old-World monkey models than in those from humans.
Conclusions
Overall, the findings provided evidence that the in vivo expression of human transgenes significantly improved xenograft survival as opposed to the inactivation of the porcine glycan genes alone.
Furthermore, the study also showed that non-human primates provided a viable model for preclinical studies to test xenotransplants.
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