Combining methylated cell-free DNA sequencing and single-cell atlases in pig-to-human xenotransplantation | LC 25

Biography

Brendan Keating, DPhil, is an Associate Professor in the Department of Surgery and The Institute of Systems Genetics at New York University Langone Health. He completed a postdoctoral fellowship at the Institute of Translational Medicine and Therapeutics with a faculty position in the Departments of Pediatrics and Surgery at the University of Pennsylvania, and he was also a Visiting Scientist at the Wellcome Trust Sanger Institute.

Brendan leads several NIH-funded transplant biomarker studies, allo-transplant-related clinical trials, and multiomic studies in NYU’s pig-to-human xenotransplant program.

Abstract

Only one in four transplant waitlisted patients receive a life-saving organ. Xenotransplantation of pig organs into humans is a promising avenue for addressing organ shortage. Understanding the immune and physiological responses in these xenotransplants is crucial for future success in compassionate use and first-in-human trials. Seven gene-edited pig organs were transplanted into brain-dead or living humans in NYU in the last three years. These studies allowed us to generate and combine a number of omic datasets, including long-read whole-genome sequencing (WGS) atlas of the human and pig, extensive transcriptomics including peripheral blood mononuclear cells (PBMC) bulk RNA-seq, tissue and PBMC sc/snRNA-seq and spatial transcriptomics, and various targeted and untargeted proteomic and metabolomic profiling. Plasma and urine samples were collected every day along with frequent xenograft tissue biopsies. Conventional donor-derived cell-free DNA (cfDNA) of acute allograft rejection in human allotransplants relies on a clinically meaningful increase in absolute or relative levels cfDNA from damage donor graft tissue. In efforts to generate prognostic and diagnostic methylated cfDNA (m-cfDNA) markers of xenograft rejection, we created a single-cell methylation WGS atlas from >18,000 nuclei in the pig kidney xenograft linked to nanopore sequenced cfDNA (n=80 timepoints). We have generated m-cfDNA profiling using these Oxford Nanopore Technologies sequencing and analyses pipelines on plasma cfDNA daily timepoints spanning dense timepoints in living and brain-dead time-courses, assessing their prognostic and diagnostic value. cfDNA profiles were also interrogated for infectious microbes.