Defining drivers of mtdna levels in blood components and their impact on transfusion-related lung injury

Vitalant Research Institute received a $262,500 Project Grant award from the National Heart, Lung, and Blood Institute (NHLBI) under the Cardiovascular Diseases Research program (CFDA 93.837), effective September 15, 2025, with completion targeted for August 31, 2027. The award funds research aimed at defining the drivers of mitochondrial DNA (mtDNA) levels in blood components and investigating their impact on transfusion-related acute lung injury (TRALI). The research will characterize the genetic association between the ANKLE1 gene and mtDNA levels in red blood cell units, identify which blood components harbor the majority of mtDNA, and evaluate the mechanistic relationship between ANKLE1 protein expression and mtDNA. Additionally, the project will develop and optimize new laboratory assays, including ELISA and flow cytometry methods, to measure ANKLE1 expression at both bulk and cellular levels across different sample types.

This research represents an advancement in precision transfusion medicine by potentially identifying novel genetic markers of blood donors that could increase transfusion-related injury risk. Should ANKLE1 protein levels prove associated with mtDNA, the findings would establish a potential druggable target for modulating mtDNA in blood products, with significant implications for enhancing transfusion safety and reducing non-infectious transfusion complications.

Abstract

Blood transfusion has proven to be a life-saving treatment for those with acute and chronic blood loss. Nonetheless, blood transfusion poses its own risks, and in recent years the dangers of transfusion above and beyond the infectious risks have gained attention. Relatively less attention has been paid to the non-infectious compared to the infectious consequences of transfusion in the time since HIV appeared as an infectious risk of transfusion. In this proposal we will explore a recently discovered genetic association underlying mtdna levels in RBC units and the potential to increase risk of acute lung injury, with implications for transfusion medicine and beyond. This proposal will explore a newly determined link between ANKLE1 and mtdna in blood donor products. The idea that a novel genetic marker of blood donors who could potentially increase the risk of acute lung injury after transfusion would represent an important step in forwarding the concept of precision transfusion medicine and enhancing transfusion safety. In addition to potentially associating ANKLE1 genetic variation with mtdna levels and acute lung injury, this proposal will define which components of blood harbor the majority of mtdna. Furthermore, linking mtdna level with ANKLE1 protein expression would provide an important mechanistic link between the gwas findings presented in our preliminary data. If ANKLE1 protein level is shown to be associated with mtdna, IT would provide a potential druggable target to affect mtdna in blood products. In addition to studying a novel topic, several new assays will be optimized for this project. ANKLE1 will be measured via elisa and flow cytometry to quantify expression at the bulk and cellular level. We will also optimize how different sample types (whole blood VS. Plasma) can be assayed, including methods to prevent hemoglobin from interfering with the assays used. This proposal will accomplish two main goals. First, we will determine if mtdna in blood products predicts subsequent acute lung injury in transfusion recipients. Finding such an association would open the door to decreasing the risk of blood transfusion by either screening blood products or developing methods to reduce mtdna levels in products and test these modified products in animal models. Second, we will determine where mtdna is most plentiful in blood products and explore the hypothesis that ANKLE1 levels will inversely correlate with mtdna burden. Understanding the regulation of mtdna would allow development of potential therapeutics to modify levels in blood products.