Design and testing of a humanized porcine donor for xenotransplantation

Recent human decedent model studies1,2 and compassionate xenograft use3 have explored the promise of porcine organs for human transplantation. To proceed to human studies, a clinically ready porcine donor must be engineered and its xenograft successfully tested in nonhuman primates. Here we describe the design, creation and long-term life-supporting function of kidney grafts from a genetically engineered porcine donor transplanted into a cynomolgus monkey model. The porcine donor was engineered to carry 69 genomic edits, eliminating glycan antigens, overexpressing human transgenes and inactivating porcine endogenous retroviruses. In vitro functional analyses showed that the edited kidney endothelial cells modulated inflammation to an extent that was indistinguishable from that of human endothelial cells, suggesting that these edited cells acquired a high level of human immune compatibility. When transplanted into cynomolgus monkeys, the kidneys with three glycan antigen knockouts alone experienced poor graft survival, whereas those with glycan antigen knockouts and human transgene expression demonstrated significantly longer survival time, suggesting the benefit of human transgene expression in vivo. These results show that preclinical studies of renal xenotransplantation could be successfully conducted in nonhuman primates and bring us closer to clinical trials of genetically engineered porcine renal grafts.


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Sample size
We did not run an a priori power calculation.Essentially, this was not done because, as the reviewer noted, NHP studies are expensive and complex and we simply do not expect to be able to perform a statistically powered study in NHP.Sample sizes among the groups in our study were chosen based on historical studies from the xenotransplantation community and what is practical.
Data exclusions Three transplants were excluded from the 3KO.7TG group.These porcine donors were cloned from nuclear donor cells of the EG114-124 edited clonal population of cells, which by NGS analysis was later found to carry a rearranged Payload 15S sequence at the AAVS1 genomic safe harbor site.Due to this complication, expression of the ssEEF1A cassette carrying the TNFAIP3 and HMOX1 genes was compromised.In consultation with Dr. George Caputa (on April 17, 2023), our editor from Nature, we made the decision not to include these 3 transplants for the 3KO.7TG group.

Replication
NHP transplantations were performed over the course of 2 years, from October, 2020, to July 2022, involving 3 academic groups (MGH, Duke, and UW Madison).The survival benefit of the 3KO.7TG+/-RI genotype is consistently achieved, as compared with the 3KO+/-RI group.For in nature portfolio | reporting summary April 2023 vitro analyses of the function of the genetic edits on endothelial cells, experiments were performed independently at least twice with similar outcomes.
Randomization The porcine donors were cloned from two clonal populations of edited cells, EG114-94 or EG114-137, and originated from the same wild type pig, Yuc25F.These two clones were confirmed to carry the 3KO.7TG+/-RI edits by extensive NGS analysis and therefore, used interchangeably.NHP recipients were chosen based on low performed antibody binding to porcine cells, in no particular order.Their assignment into one group over the other is presented in Extended Data Figure 6.The 3KO.7TG porcine donors were the first to come off our production line and their kidneys were transplanted into the NHPs in between 2020 and 2021.Next off the production line were the 3KO.7TG.RI donors, whose kidneys were transplanted between 2021 and 2022.The 3KO+/-RI genotypes were transplanted in 2022.For in vitro studies to investigate the function of the genetic edits on primary endothelial cells, endothelial cells used in these experiments from multiple porcine donors were chosen randomly based on the number of frozen vials we had in our possession.Once we decided on the donors to use,.all the in vitro functional analysis with KECs was performed with cells from the same donors across the different assays.For the studies with AECs, AECs were chosen based on what we had stored in liquid nitrogen.

Blinding
The 3 academic centers (MGH, Duke, and UW Madison) performed the NHP transplantation studies and were not blinded about the genotypes of the porcine donors.We did not believe it was necessary, as it was obvious that the 3KO+/-RI group uniformly did not survive well.For in vitro studies, samples used in the functional analyses were not blinded as we knew the genotypes of the porcine donor cells used in the assays and we knew which NHP recipient (and their porcine donor identity) serum/blood samples were being analyzed.
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