Targeting brain myeloid cells with siRNA-lipid conjugates for treatment of Alzheimer's disease - Summary Brain myeloid cells, including microglia and macrophages, selectively express several genes that confer risk for late-onset Alzheimer’s disease (AD)—for example CD33, which regulates amyloid beta clearance. As such, there is substantial interest in using oligonucleotide drugs—including siRNA—to knock down genes such as CD33 in brain myeloid cells as a therapeutic intervention. However, it remains challenging to deliver siRNA to these key cells in the brain. Oligonucleotide drugs are typically administered directly into cerebrospinal fluid (CSF), but free siRNA is cleared rapidly and exhibits poor cellular uptake. To combat this issue, conjugation to lipophilic moieties like cholesterol has been explored as a strategy to improve siRNA tissue retention and knockdown activity. Unfortunately, current lipid modifications can impart dose-limiting toxicity and generate steep concentration gradients around the injection site that cannot necessarily reach distal brain structures by diffusive transport. In prior work, we have developed a novel diacyl fatty acid carrier for siRNA (“siRNA-EG18”) that binds albumin with high affinity to extend circulation half-life of the conjugated siRNA. In the brain, albumin is transported effectively along perivascular spaces that reach deep brain structures, leading us to hypothesize that siRNA-EG18 could leverage convective perivascular transport to improve targeting of myeloid cells throughout the entire brain after delivery into CSF. Our extensive preliminary and supporting data support this premise, where we have demonstrated the ability of siRNA-EG18 to distribute through perivascular spaces and silence genes in brain myeloid cells using both bulk and single-cell assays. Moving forward, Aim 1 will comprehensively analyze siRNA- EG18-mediated bulk gene and protein silencing activity in brain myeloid cells as a function of injected dose. Aim 2 will extend these analyses into contexts of aging and amyloidosis and incorporate single-cell sequencing to understand siRNA-EG18’s ability to target myeloid cell subtypes under disease-related conditions. Aim 3 will focus on translation by benchmarking the efficacy of siRNA-EG18 against a lipid-siRNA conjugate that has entered phase 1 clinical trials for early-onset AD. All aims will target Cd33 for its connection to myeloid cells and late-onset AD. Successful completion of this work will establish siRNA-EG18 as a promising technology for AD treatment through myeloid cell gene targeting.