Neutralizing Circulating Histones With an RNA Aptamer to Prevent MultiorganDysfunction in Sepsis

Every year, sepsis affects more than 30 million people and is the primary cause of morbidity and mortality in noncoronary intensive care units in the Western world. In the Veterans Affairs healthcare system, sepsis hospitalizes over 35,000 Veterans annually. Patients with severe sepsis have mortality rates of 33% at 28 days and 71% at three years. The management of sepsis is mainly supportive while waiting for antibiotic therapy to be effective. Recent evidence suggests that the molecular mechanisms responsible for organ dysfunction associated with sepsis involve the release of nuclear proteins, of which histone proteins are the most abundant. The applicant’s laboratory recently developed RNA aptamers that bind with high affinity and specificity to human histones but not to other serum proteins. Administration of this selected RNA aptamer, referred to as KU7, inhibited histone-induced platelet aggregation, Toll-like receptor activation, endothelial cell death, and calcium influx in cultured cells. Preliminary data showed that KU7 protects against weight loss and pulmonary edema and improves the survival of mice challenged with lipopolysaccharide endotoxins. As a next step, studies are needed to test the efficacy of KU7 in an animal model of sepsis. The data derived from this project will provide the necessary information for a competitive application for additional grant funding. Experiments will test the hypothesis that the RNA aptamer KU7 reduces organ dysfunction and improves survival in a murine model of sepsis. Aim 1 will establish the cecal slurry (CS) model of sepsis in mice in the applicant’s laboratory. The severity of sepsis will be titrated to allow subsequent experiments to study illness severity and mortality. Serum histone levels will be measured at different time points after the CS injection to identify the optimal timing of the initial dose of KU7. Aim 2 will determine the efficacy of KU7 in improving survival and illness severity in sepsis. As sepsis is associated with the release of bacterial nucleases, the effectiveness of two different chemical modifications of KU7 will be compared. These studies will also identify the minimum and maximum effective dose of KU7, determine whether KU7 provides additional benefits to antibiotic therapy, and compare KU7 with a previously described neutralizing histone antibody. Aim 3 will perform a series of biochemical and histological analyses to assess the efficacy of KU7 in protecting against organ dysfunction in sepsis. The significance of this project is supported by the prevalence and complications of sepsis and the absence of FDA-approved therapies to prevent histone-mediated tissue injury. The innovative strength of this proposal is the identification of novel therapeutic bioreagents that, if successful, would change the paradigm of treatment of critically ill patients with sepsis. As therapeutics, aptamers will not have the same limitations as other biologics and will provide rapid and easy delivery with the potential to save lives.