Abstract A073: Engineered Exosomes Carrying Rabies Viral Glycoprotein (RVG) and Neuroglobin (Ngb) Improved Stroke Outcome in Mouse Model of tMCAo.

Delivering therapeutic agents to the central nervous system (CNS) is a significant challenge due to the BBB. To overcome this obstacle, researchers have used the rabies virus glycoprotein (RVG) that is found on the rabies virus envelope, which plays a crucial role in this process. The 189–214 amino acid region of RVG is essential for binding to nerve cells and enabling viral entry and fusion. In 2000, neuroglobin (Ngb), the third member of the globin family, was identified in the brains of humans and mice. Both in vitro and in vivo studies have shown that Ngb protects the brain from hypoxic, ischemic, and oxidative stress through its ability to bind oxygen reversibly. Recently, our laboratory has developed a DNA technology-based platform to make engineered exosomes using non-tumorous HEK293 cells that carry and express specific cell-targeting peptides (RGV) as well as therapeutic probes (Ngb). In this study, we have employed engineered exosomes carrying both RVG and Ngb as targeting and therapeutic agents to improve the outcome in tMCAo stroke in 12-16-month-old mice. We stipulate that engineered exosomes carrying neuron-specific RVG peptides as targeting ligands and therapeutic probe, Ngb , to protect neurons from hypoxic/ischemic and oxidative stress-related insults following ischemic stroke can be delivered to the injured neurons in the stroke and peri-infarct areas to improve the stroke outcome. We have successfully developed the engineered exosomes and proven the accumulation of the exosomes in the neurons in the stroke areas. Despite similar hypoperfusion on laser speckle images, magnetic resonance imaging (MRI) showed significantly smaller stroke volume in animals (n=20) that received engineered exosomes carrying both RVG and Ngb compared to the control exosomes and exosomes carrying gon ly RVG and Ngb separately. RNA seq, western blot, and proteomics data showed higher expression of Ngb, SOD, and NADH, indicating the protection of cells from oxidative stress and activation of the electron transport chain in mitochondria, which indicates an active role in cellular respiration, respectively. Immunohistochemistry showed a higher number of blood vessel formations in animals treated with exosomes carrying both RVG and Ngb. Despite the worst neurodeficit score on day 7, animals treated with exosomes carrying both proteins showed better recovery on day 28. Engineered exosomes carrying both RVG and Ngb could be an agent for treating stroke.