Malong Liza, Roskosch Jessica, Hager Carolina, Fortin Jean-Philippe, Schmucki Roland, Callow Marinella G, Weile Christian, Romeo Valentina, Patsch Christoph, Martin Scott, Costa Mike, Modrusan Zora, Villaseñor Roberto, Koller Erich, Haley Benjamin, Spang Anne, Roudnicky Filip
Therapeutic Modalities, Pharmaceutical Research and Early Development, F. Hoffmann-La Roche Ltd, Basel, Switzerland.
Department of Bioinformatics and Computational Biology, Genentech, Inc., San Francisco, CA, USA.
Nat Commun. 2025 Jun 30;16(1):5378. doi: 10.1038/s41467-025-61039-y.
Anti-sense oligonucleotides (ASOs) are modified synthetic single-stranded molecules with enhanced stability, activity, and bioavailability. They associate with RNA through sequence complementarity and can reduce or alter mRNA expression upon binding of splice site positions. To target RNA in the nucleus or cytoplasm, ASOs must cross membranes, a poorly understood process. We performed an unbiased CRISPR/Cas9 knockout screen with a genetic splice reporter to identify genes that can increase or decrease ASO activity, resulting in the most comprehensive catalog of ASO-activity modifier genes. Here we reveal distinct targets, including AP1M1 and TBC1D23, linking ASO activity to transport of cargo between the Golgi and endosomes. AP1M1 absence strongly increases ASO activity by delaying endosome-to-lysosome transport in vitro and in vivo. Prolonged ASO residence time in the endosomal system may increase the likelihood of ASO escape. This insight into AP1M1 role in ASO trafficking suggests a way for enhancing the therapeutic efficacy of ASOs by manipulating the endolysosomal pathways.
反义寡核苷酸（ASO）是经过修饰的合成单链分子，具有更高的稳定性、活性和生物利用度。它们通过序列互补与RNA结合，并在结合剪接位点位置后能够降低或改变mRNA表达。为了靶向细胞核或细胞质中的RNA，ASO必须穿过细胞膜，这是一个尚未完全了解的过程。我们使用基因剪接报告基因进行了无偏向性的CRISPR/Cas9基因敲除筛选，以鉴定能够增加或降低ASO活性的基因，从而得到了最全面的ASO活性修饰基因目录。在此，我们揭示了不同的靶点，包括AP1M1和TBC1D23，它们将ASO活性与高尔基体和内体之间的货物运输联系起来。在体外和体内，AP1M1缺失通过延迟内体到溶酶体的运输，强烈增加了ASO活性。ASO在内体系统中的停留时间延长可能会增加ASO逃逸的可能性。对AP1M1在ASO运输中作用的这一见解，为通过操纵内溶酶体途径提高ASO的治疗效果提供了一种方法。
