Principal Investigator/Program Director (Last, First, Middle: Soon Seog Jeong Abstract Due to its narrow time-window of administration (up to 4.5 hours post symptoms) and 6-7 fold increased risk of intracranial hemorrhage, merely 3-5% of acute ischemic stroke patients receive recombinant tissue plasminogen activator (tPA), the only FDA-approved drug for this indication. NGF prevents neuronal apoptosis in primary cultured neurons and reduces neuronal degeneration in animal models of neurodegenerative diseases. In the central nervous system, NGF is produced throughout adult life and primarily targets basal forebrain and striatal neurons. It has been shown that intranasal (IN) NGF bypassed the blood-brain barrier and distributed in the whole brain without peripheral adverse effects. IN administration of wildtype NGF significantly reduced infarct volume and improved neurological outcomes by protecting neurons from ischemic injury, promoting angiogenesis, and enhancing striatal neurogenesis with at least a 24h treatment window after stroke onset. We have optimized a human NGF variant and developed a cost-effective protein production system for making the variant. This proprietary variant selectively activates the NGF TrkA receptor with enhanced activity to promote neuron survival and function. In a randomized and blinded study in rats, IN treatment with the variant for 3 weeks 6h after thrombo-embolic stroke (outside the therapeutic treatment time window for rt-PA) robustly improved short- and long-term neurological deficit score as assessed 28 days after stroke. In this study, we will follow the STAIR recommendations and RIGOR guidelines to conduct a Phase I preclinical study to validate the the combination treatment with tPA in a rat model of stroke using both male and female animals. The long-term goal is to develop the dug candidate as acute or sub-acute therapy, alone or in combination with tPA, to safely reduce disability for millions of stroke patients. Specific Aim. Determine whether combining tPA with the proprietary NGF variant more effectively improves long-term outcomes compared to vehicle and tPA in a rat model of thrombo-embolic stroke when administered at 6h after occlusion.