Intratracheally administered LNA gapmer antisense oligonucleotides induce robust gene silencing in mouse lung fibroblasts

The lung is a complex organ consisting of various cell types, including leukocytes, endothelial cells, epithelial cells, and fibroblasts, each of which has distinct roles in normal and disease contexts. Antisense oligonucleotides (ASOs) have been studied in the lung, but it has been a challenge to determine their effectiveness in each cell type due to the lack of appropriate analysis methods. In this work, we employed three distinct approaches to study silencing efficacy within different lung cell types. First, we reasoned that silencing of a cell-surface protein could be used to demonstrate the degree of silencing in different cell populations directly in a flow cytometry experiment. As such, we used cell lineage markers to identify specific types, and measured silencing of cell-surface proteins CD47 or CD98 after treatment with LNA gapmer ASO. Lung fibroblasts were the most susceptible cell type to locally delivered ASOs, with significant silencing also seen in endothelial cells. Second, we applied single-cell RNA sequencing (scRNA-seq) to measure the silencing efficacy in distinct cell types; to the best of our knowledge this is the first time scRNA-seq has been applied to measure the efficacy of oligonucleotide therapeutics. In scRNA-seq, fibroblasts and endothelial cells showed high ASO-mediated silencing of Malat1. Third, we confirmed that the robust silencing in lung fibroblasts is broadly applicable by silencing two targets expressed mainly in fibroblasts, Mfap4 and Adam33. Thus, across all three independent approaches, our results demonstrate that intratracheally administered LNA gapmer ASOs robustly induce gene silencing in lung fibroblasts. ASO-induced gene silencing in fibroblasts was durable, lasting 4-8 weeks after a single dose. Thus, lung fibroblasts are well aligned with ASOs as therapeutics.