Cell-type-specific Evaluation of Liver Allografts during Normothermic-Machine-Perfusion

Liver transplant (LTx) is the standard-of-care for patients with end-stage liver disease. While in 2023 a record number of deceased donor liver transplants were performed, the demand for liver transplants continues to exceed the availability of suitable donor organs. Three key challenges hinder the utilization of potential donor livers: (1) logistical constraints due to short timeframes allowed by static cold storage, (2) concerns about organ quality and complications from marginal organs, and (3) injury from ischemia-reperfusion (IRI). The recently FDA-approved Normothermic Machine Perfusion (NMP) technology has been a key breakthrough for LTx as it can address all three aforementioned challenges: (1) NMP can preserve the donor organ in a functional state for up to 12-24 hours; (2) high-level viability parameters such as vascular flow, bile production, as well as glucose and lactate metabolization allow for a functional assessment of organ quality; and (3) ischemic injury in donor organs can be ameliorated by minimizing further injury during transport and storage. While NMP has been a gamechanger for LTx on its own, coupling it with another recent breakthrough – cell-free methylated DNA (cfDNA) monitoring – could create an even better clinical decision-making framework as well as more favorable patient outcomes. Specifically, analysis of cfDNA fragments shed by dying cells can reveal allograft as well as host tissue damages at the cellular level by mapping methylation patterns of individual cfDNA molecules to tissue- and cell-type specific markers. This could be especially pivotal in LTx given the multitude of cellular compartments in the liver and their related complications. Our proposed study hypothesizes that cfDNA-derived cell type-specific injury markers can serve as novel viability parameters to assess the quality and viability of donor livers during NMP, which we will study via two aims: AIM #1: To establish cfDNA-derived cell type-specific injury markers as novel biomarkers for determining organ viability at the time of machine perfusion. AIM #2: To establish cfDNA-derived cell type-specific injury markers as novel biomarkers for determining the nature and severity of complications post-transplant. Our team has access to previously collected samples from 91 livers that underwent NMP at our center, and we have already established the required methods, including our recently expanded DNA methylation atlas that includes hepatocytes, hepatic stellate, endothelial, and resident immune cells as well as biliary epithelia. This project can pave the way for establishing more efficacious methods to assess via biomarkers whether a liver that is undergoing NMP is viable for transplant, as well as which specific complications from cellular damage arise once transplanted. It thus has the potential to expand the number of livers that get transplanted, enhance monitoring thereof, and inform therapeutic strategies post-transplant, all of which can help bring this life-changing intervention to more patients currently awaiting a liver transplant. Project Number: 1R21AI191090-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Anton Wellstein (+1 co-PI) | Institution: GEORGETOWN UNIVERSITY, WASHINGTON, DC | Award Amount: $429,000 | Activity Code: R21 | Study Section: Hepatobiliary Pathophysiology Study Section[HBPP] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21AI19109001