2022 Ono Pharma Foundation Symposium

Title: Development of next-generation antisense oligonucleotides for potent gene silencing in the lung

Jonathan Watts
Associate Professor
RNA Therapeutics Institute
UMass Medical School

Abstract: Oligonucleotide therapeutics have not yet found significant success in the lung, a complex organ with distinct cell types. In the first part of this talk, we describe the silencing efficacy of antisense oligonucleotides (ASOs) in different cell types of the lung. First, we used lineage markers to identify cell types by flow cytometry, and simultaneously measured ASO-induced silencing of cell-surface proteins. Second, we applied single-cell RNA sequencing (scRNA-seq) to measure 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 both approaches, fibroblasts were the most susceptible to locally delivered ASOs, with significant silencing also in endothelial cells. We confirmed that the robust silencing in fibroblasts is broadly applicable by silencing targets expressed mainly in fibroblasts. ASO-induced gene silencing in fibroblasts was durable, lasting 4-8 weeks after a single dose. The second part of the talk demonstrates that divalent asymmetric siRNAs (di-siRNAs) are bioavailable and active in the lung after local administration. Through the systematic screening of hundreds of oligonucleotide sequences, we identified fully chemically stabilized siRNAs and ASOs that target regions of the SARS-CoV-2 genome conserved in all variants of concern, including delta and omicron. We successively evaluated candidates in cellular reporter assays, followed by viral inhibition in cell culture, with eventual testing of leads for in vivo antiviral activity in the lung. The optimized divalent siRNAs showed robust antiviral activity in human cells and mouse models of SARS-CoV-2 infection. Di-siRNAs and advanced ASOs are a promising tool for genetic and fibrotic lung diseases as well as for current and future respiratory viral pandemics.