The lung is a complex organ with various cell types having distinct roles. Antisense oligonucleotides (ASOs) have been studied in the lung, but it has been challenging to determine their effectiveness in each cell type due to the lack of appropriate analytical methods. We employed three distinct approaches to study silencing efficacy within different cell types. First, we used lineage markers to identify cell types in flow cytometry, and simultaneously measured ASO-induced silencing of cell-surface proteins CD47 or CD98. 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. Third, we confirmed that the robust silencing in fibroblasts is broadly applicable by silencing two targets expressed mainly in fibroblasts, Mfap4 and Adam33. Across independent approaches, we 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.

中文翻译：

---

气管内施用的 LNA gapmer 反义寡核苷酸在小鼠肺成纤维细胞中诱导强大的基因沉默

肺是一个复杂的器官，各种细胞类型具有不同的作用。已经在肺中研究了反义寡核苷酸 (ASO)，但由于缺乏适当的分析方法，很难确定它们在每种细胞类型中的有效性。我们采用三种不同的方法来研究不同细胞类型中的沉默功效。首先，我们在流式细胞术中使用谱系标记来识别细胞类型，同时测量 ASO 诱导的细胞表面蛋白 CD47 或 CD98 的沉默。其次，我们应用单细胞 RNA 测序 (scRNA-seq) 来测量不同细胞类型的沉默效果；据我们所知，这是首次将 scRNA-seq 应用于测量寡核苷酸疗法的疗效。在这两种方法中，成纤维细胞对局部递送的 ASO 最敏感，在内皮细胞中也有显着的沉默。第三，我们通过沉默主要在成纤维细胞中表达的两个靶点 Mfap4 和 Adam33 证实了成纤维细胞中的稳健沉默是广泛适用的。通过独立的方法，我们证明气管内施用的 LNA gapmer ASO 可强烈诱导肺成纤维细胞中的基因沉默。ASO 诱导的成纤维细胞基因沉默是持久的，单次给药后持续 4-8 周。因此，肺成纤维细胞与作为治疗剂的 ASO 非常吻合。我们证明了气管内施用的 LNA gapmer ASO 可强烈诱导肺成纤维细胞中的基因沉默。ASO 诱导的成纤维细胞基因沉默是持久的，单次给药后持续 4-8 周。因此，肺成纤维细胞与作为治疗剂的 ASO 非常吻合。我们证明了气管内施用的 LNA gapmer ASO 可强烈诱导肺成纤维细胞中的基因沉默。ASO 诱导的成纤维细胞基因沉默是持久的，单次给药后持续 4-8 周。因此，肺成纤维细胞与作为治疗剂的 ASO 非常吻合。