Abstract of proposed Alzheimer’s disease-related supplement In 2021, Alzheimer’s disease (AD) affects more than 20 million people worldwide, with about 135 million people expected to develop AD by 2050 and 3.3% of the US population by 2060. Our goal in this supplement is to apply the positron emission tomography (PET) tools developed in the parent grant to the assessment of gene delivery in AD. Adeno-associated viruses (AAVs) are a promising tool and can be used to encode for growth factors, hormones, Cdk5 inhibitors, transcription factors, immune factors, modulators of Aβ metabolism, tumor necrosis factor, synaptic function mediators, neural function mediators, or age suppressing factors in the treatment of AD. In addition, AAVs have the potential to enable gene editing. Here, we specifically will deliver genes encoding reporters or brain-derived neurotropic factor (BDNF) in an AAV. BDNF was selected for this proof of concept study due to the first-in-human study in AD (NCT05040217) that has recently begun based on stereotactic AAV injection. Delivery of gene and protein therapy to the brain has traditionally been extremely limited but may be further limited in AD. Autoimmune neurological diseases are often linked to a dysfunctional BBB. With neuroinflammation, reactive oxygen species (ROS) generation can disrupt the BBB via various mechanisms. BBB dysfunction is hypothesized to play a role in AD pathology as a result of impaired P- glycoprotein mediated efflux from the brain or reduced LRP1 expression and resulting reduced transport of Aβ across the BBB. Using a directed evolution approach to viral capsid engineering and selection, the Gradinaru group at Caltech identified specific peptides that, when displayed on the surface of modified capsids, enhanced BBB transport and neuronal transduction compared to the conventionally-used adeno-associated virus AAV9, following intravenous (IV) injection in mice. We developed novel combined positron emission tomography (PET) imaging techniques that non-invasively assess the pharmacokinetics of the AAV over the first days after injection and the resulting gene expression over months or potentially years. For gene transduction reporting, we include the pyruvate kinase M2 (PKM2) reporter gene which has a low background level in the brain and is imaged with [18F]DASA-23, a tracer that freely crosses the BBB. The brain uptake of these novel AAVs studied here by PET imaging reaches an extraordinary temporal-peak spatial-maximum of ~35% ID/cc at 4 h post- injection in mouse models. Recent selections at Caltech have provided additional capsids that transduce the mouse and primate brain with reduced expression in the liver, spleen, kidneys, and lungs. Our resulting specific aims are to: 1. Validate and apply PET imaging techniques to assess brain delivery of a radioactive tag and reporter gene in mouse models of AD using both traditional and novel AAV capsids, and 2. Evaluate BDNF protein production as a result o...