ABSTRACT: This application focuses on the development of novel vasculogenic cell therapies to treat cerebral blood flow (CBF) deficits in Alzheimer’s disease (AD). The goal is to improve CBF in AD by pre-programming fibroblasts to convert into induced endothelial cells (iECs) to drive the formation of new vascular tissue, in the brain, and stem the progression of AD, which is the most common form of dementia, currently affecting millions of people worldwide. Currently, there is no available therapy for AD, and treatments are mainly focused on managing symptoms. Numerous studies show a clear link between CBF deficits and AD in patients and murine models of AD. CBF deficits precede the formation of amyloid beta plaques and phosphorylated tau tangles, as well as the cognitive decline associated with AD. Moreover, recent studies in murine models of AD show that pharmacological strategies aimed at increasing CBF lead to a rapid improvement in behavioral deficits. Taken together, these observations suggest that impaired CBF plays a key role in AD, and that increasing CBF could be a viable strategy to treat or prevent AD. Under my New Innovator Award (DP2), we have been working on the development nanotransfection-based (i.e., non-viral), reprogramming-driven cell therapies for ischemic stroke. Recently we reported on a novel reprogramming-based vasculogenic cell therapy for the treatment of ischemic stroke. In this study we showed that fibroblasts that have been pre-programmed to convert into iECs, in vivo, can improve cortical perfusion in mice that have suffered an ischemic stroke, enhance stroke recovery, and reduce motor impairment compared to mice treated with control fibroblasts. iECs can be derived from fibroblasts via nanotransfection of transcription factor genes, ETV2, FOXC2, and FLI1 (EFF). Nanotransfected cells can also release extracellular vesicles (EVs) loaded EFF, which have potent vasculogenic/angiogenic effects, and can help to amplify iEC-directed conversions. Promising pilot studies run in response to the exciting stroke data support the scientific premise that fibroblasts pre-programmed to generate iECs can also increase CBF and reduce cognitive deficits in a mouse model of AD. Here we are proposing to build upon these striking observations to study, for the first time, whether iEC-directed reprogramming of fibroblasts, and/or EFF-loaded EVs, can be a viable strategy to counteract CBF deficits and pathology progression under AD. Currently, there is a paucity of research on vasculogenic cell or EV therapies aimed at addressing CBF deficits to prevent, delay, or reverse AD. As such, the work proposed herein is highly innovative and potentially transformative. Upon completion, we expect to have rigorously demonstrated the benefits of EFF-nanotransfected fibroblasts or EFF- loaded EVs in the prevention/treatment of AD. Outcomes from this study will be leveraged to pursue future studies on the safety and efficacy of nanotransfection-driv...