Project 2

New York University School Of Medicine, New York NY

Investigators

Abstract

Project Abstract: U19 Project 2 Cell-free DNA and Protein Wet-lab and Analyses in Pig to human Xenotransplantation Xenotransplantation (XTx) of solid organs from domesticated pigs serves promising in mitigating the critical organ shortages, with only ~25% of the waitlisted individuals receiving an allograft. Although recent advancements including GGTA1 pig knockouts and FDA-approved GalSafeTM pigs have demonstrated efficacy in overcoming hyperacute antibody-mediated rejection and extended survival in non-human primates, robust and reliable biomarkers for monitoring xenograft cellular health and rejection are essential to progress to clinical trials and usage in living humans. Current minimally invasive human allograft surveillance tests rely primarily on donor-derived cell-free DNA (dd-cfDNA) which detects increases in cell-free DNA from damaged donor allograft cells in the recipient plasma. However, methylated-dd-cfDNA (m-dd-cfDNA) offers increased specificity as distinct methylation patterns make it feasible to distinguish cell-type-specific DNA from the xenograft. This may be important in the context of defining the specific injury to a xenograft as early waves of antibody-mediated rejection (AbMR) damage specific cell-types such as endothelial cells. Using a combination of cfDNA and m-dd-cfDNA plasma patterns from pig-to-human decedent XTx studies and pig kidney, liver, and heart xenograft atlases of major cell types using single-nucleus methylation whole-genome sequencing (m-snWGS), our study aims is to develop prognostic and diagnostic markers of pig xenograft damage, including AbMR and acute cellular rejection (ACR). Preliminary data from a 61-day pig kidney xenotransplant study performed in NYU revealed a ~6-fold increase in pig kidney cfDNA during AbMR. In the 61-day study, the end-of-study pig kidney xenograft biopsy was used to generate a cell atlas from 18,400 single nuclei. The nuclei were subjected to m-snWGS, revealing clear methylation differences for 14 major pig kidney cell types. Methylated-dd-cfDNA data was generated in 13 timepoints across the 61-d study, and the methylated patterns were mapped back to the pig kidney cell atlas. As there was a biopsy proven rejection event in the middle of the 61-day study (as evident from conventional histological damage and in situ transcriptomics), we selected several timepoints around this key event. Elevation of endothelial cell m-cf-ddDNA markers prior to xenograft cellular damage were found. We specifically aim to perform nanopore sequencing of human recipients of pig xenografts. Further, in pig xenografts, we propose to generate methylated single-nucleus whole-genome sequencing atlases followed by their comparison with m-dd-cfDNA plasma of the recipients. We will also analyze cell-free pig protein patterns from blood and compare them with cfDNA from equivalent timepoints. These efforts will ultimately improve the xenograft survival and function by identifying specific cfDNA and protein diagnostic and prognostic signatures in the plasma indicative of specific cellular damage in the xenograft.