Xenotransplantation literature update, September—October 2015 (2015)

Xenotransplantation success depends on understanding the impact of endogenous viruses on xenograft survival. Dr. Jochim Denner has provided a valuable review of porcine cytomegalovirus (PCMV), similarities to human CMV (HCMV)-associated transplant rejection and cautionary advice on how to proceed with proper screening methods 1. Cytomegalovirus infection is a significant concern for transplant success, regardless of host. Despite expected restriction in humans, PCMV was recently shown to infect human cells in culture. The importance of this finding is highlighted by multiple xenotransplantation trials using non-human primates where the presence of PCMV in xenografts was linked with decreased transplant survival. Thus, risk management for all xenotransplantations should include screening for CMV species in both donor and recipient. The author suggests that management of PCMV must begin by eliminating the virus from infected herds using a combination of screening, breeding, and drug treatment approaches. Piglets become infected in utero and thus delivery of piglets via Cesarean section into pathogen-free facilities can reduce transmission. Importantly, continued surveillance using sensitive detection methods of porcine donors akin to that of human donors is necessary prior to xenotransplantation using porcine tissues. Porcine endogenous retrovirus (PERV) is present in numerous copies within every pig genome. Although transmission of PERV in vitro is a rare event and no link to human transmission has been demonstrated, it is unclear how PERV genome contamination impacts xenotransplantation. To determine whether porcine cells could be “cured” of integrated PERV, Yang et al. 2 designed CRISPR/Cas9 oligos targeting the PERV polymerase gene (pol). Deletion of the viral polymerase would theoretically prevent virus replication. Using the CRISPR/Cas9 approach overcomes the significant hurdle to PERV elimination that the genome is present in many copies within the cell. The authors first identified that there were 62 copies of PERV in the kidney cell line being used. Of note, a single transfection event resulted in mutations in all 62 copies of the pol gene. Thus, PERV eradication in cells to be used for xenotransplantation can be facilitated by technology where the targeting guide RNA is not tethered to the nuclease cutting the target site. Genetic disruption of PERV and other potential zoonotic pathogens may create cells or organs safer for xenotransplantation if a clear risk of infection exists. The question of can we use porcine islets to cure type 1 diabetes may be trumped by the question of should we use them. In the context of islet xenotransplatnation, Ellis and Korbutt 3 have put forth a discussion of the available data supporting the scientific and ethical use of pig cells clinically. The authors point out several issues with the application of porcine cells within the hospital setting and describe technologies that can prolong islet survival in vivo. The historical summary of successful islet xenotransplantation seems to provide an impressive justification for clinical application and the potential impact on patients lives. This review also serves as a point of discussion to review our community criteria for producing pigs for clinical studies. Ramm et al. 4 have explored an important issue in the creation of sensitive and accurate in vitro assays: contamination of cells with bovine serum glycoproteins. This is an issue that many xenotransplantation researchers have observed and unofficially may contribute 5–15% of background in flow cytometery or ELISAs. Running the appropriate controls may subtract the “background” created by serum incubation at the expense of a limited dynamic range. The authors have tested this theory of contaminating glycoproteins by growing human cells, devoid of aGal, in the presence of bovine serum as compared to pig cells, with abundant aGal. A criticism of this study is the capacity to uptake serum glycoproteins and perhaps a better design would have been to include GGTA1 knockout cells in the same conditions. The authors explain how human cells incubated with human serum bind human antibodies, perhaps via blood group antigens or Fc receptor-bound antibody. However, the human cells incubated with bovine serum do not bind human antibodies or show any background binding. Perhaps the bovine serum has “blocked” sites that antibody bound to in the other experimental condition. To the authors credit they detail how their data is in contrast to several other groups including our own current and past experience with human and pig kidney and liver endothelial cells and islets. This is of particular interest to those groups who seek to culture pig cells or tissues in a cost-effective way while not increasing the immunoreactivity or obscuring the analysis of genetically engineered pigs. Krishnan et al. 5 have an interest in isolating the most and highest quality islets from the pig pancreas. The “hows” and “whens” have been a focal point of islet xenotransplantation discussions. The authors hypothesize that pre-weaned juvenile pig islets may be protected from enzyme-induced isolation damage because exocrine tissue within the pancreas may not be fully developed. Biopsies from neonatal, pre-weaned, weaned, and young adult piglets were analyzed for enzyme-based fluorescence and exocrine granule density. Neonatal and pre-weaned had the lowest developed exocrine function but unfortunately had the smallest islets, insulin production, and islet density. The authors conclude that lower exocrine enzyme function in juvenile pigs may be an advantage for protecting islets during isolation and culture. The developing fetus is immunologically incompetent and is unable to educate itself to foreign antigens. Navarro Alvarez et al. 6 propose that introducing pig bone marrow cells into the mid gestation phase of fetal development may induce tolerance as the immune system develops. To test their hypothesis, the authors injected T-cell-depleted bone marrow cells into nine fetuses. Approximately 2 months after birth, piglets challenged with xenogenic donor cells produced no antibody. Challenge with cells from a different xeno or allo donor broke the donor-specific tolerance. The authors have presented a detailed analysis of the antibody response to post-challenge and succeeded in establishing donor-specific non-responsiveness. Immune cell analysis and further investigation of routes of subsequent challenges may shed light on how tolerance was achieved. Several gene editing tools can be used to sever DNA and facilitate a natural insertion or deletion upon DNA repair. Beaton et al. 7 have brought an increased level of specificity to the repair mechanism by co-introducing a DNA template by which to repair the cut DNA. The repair template contains a mutated version of the gene at or near the cut site. The regions flanking the target area give specificity to the repair template. Beaton et al. ask how short or long the flanking regions of the repair template can be to yield the highest DNA modification efficiency. Interestingly, the donor DNA did not direct the incorporation of a biallelic nucleotide change as expected but still gave rise to GGTA1 null alleles. The authors generated a new line of genetically engineered pigs in the Minnesota mini pig background that will likely be very useful to xenotransplantation. It is unclear, however, how that was accomplished under their experimental conditions and to what effect the length of homologous arms influenced their success given the statistical variation between groups. The authors speculate that the presence of donor DNA may have enhanced NHEJ in contrast to homology directed repair. It is easy to imagine that highly specific DNA fragments could bind to the DNA and influence ligation or repair in a way that does not include the mutated template but does allow indel formation. Dr Beaton and his colleagues are making great strides in the application of new genetic technologies to large animal models and understanding how these systems can be used to benefit xenotransplantation in unimaginable ways. Endothelial cell damage during xenoperfusion can reduce the graft's ability to prevent unwarranted coagulation and impaired vasodilation. This is especially true in lung models where endothelial cell damage can prevent the secretion of von Willebrand factor and release of nitric oxide. Park et al. 8 proposed that the addition of a nitric oxide release chemical, SIN (1, 3-morpholinosydnonimine), could reduce or prevent injury during porcine lung perfusion with whole human blood. Using an ex vivo perfusion model, the authors succeeded in reducing vascular resistance and complement activation which enhanced graft function. The addition of SIN did not ameliorate the humoral attack on pig endothelial cells but may have a significantly improved overall graft survival time. Improved survival time may provide future studies the opportunity to block the rapid immune assault on lung endothelial cells.