Project Summary/Abstract Nonalcoholic steatohepatitis (NASH) is dubbed the ?stealth epidemic? and in the U.S. alone, 20 percent of the 16 million people who are estimated to have NASH will progress and develop life-threatening conditions including fibrosis, cirrhosis, hepatocellular carcinoma, and liver failure. By the year 2020, NASH is projected to be the leading indication for liver transplantation surpassing hepatitis C virus (HCV) and alcoholic related cirrhosis, and by 2025, the total NASH pharmaceutical market size is estimated to reach $15 billion. Currently, the liver biopsy is the ?gold standard? for diagnosing and monitoring NASH, yet it is invasive, expensive (~$3,000), and associated with significant patient morbidities. Importantly, a single biopsy provides only an instantaneous `snapshot' of tissue architecture and is unable to identify NASH patients who are `rapid progressors' and thus urgently require treatment, or those on therapy that are `responders'. As a result of these limitations, liver biopsy remains the key bottleneck in the safe, accurate diagnosis and monitoring of NASH. This proposal aims to develop the Glympse liver test (GLT), a noninvasive urine diagnostic that detects the activity of hepatic proteases that drive NASH. GLT consists of an injectable panel of nanosensors that detect proteases involved with inflammation and fibrosis, and produce signals that are detected in urine. The detection signals are amplified by enzymatic action in the liver and then concentrated in the patient's urine for quantification by mass spectrometry. Because enzyme activity is measurable at the molecular level before microscopic changes are evident by biopsy, the test is thus both ultrasensitive and predictive. We will demonstrate the feasibility of formulating GLT nanosensors on an inert polymer scaffold to sense matrix metalloproteinases (MMPs) ? a key family of enzymes that are involved in NASH progression and regression. We will then validate the ability of GLT to predict disease progression and regression in a mouse model of NASH, and conduct preclinical toxicology studies in preparation for an IND submission. The GLT will offer clinicians a rapid determination of the state and trajectory of the patient's liver without the morbidity of biopsy, reduce cost of diagnosis from $3,000 to $500, and raise sensitivity by 40%. The benefit to the healthcare system per patient is estimated to be $48,000 in reduced biopsy-associated costs and $145,000 in drug costs over a patient's lifetime, saving more than $12 billion annually for the 20% of NASH patients with progressive disease alone. The impact of the adoption of GLT will reach across multiple constituencies ? increasing patient care and comfort, improving physician decision making, accelerating pharmaceutical drug trials, and lowering healthcare costs.