Abstract 5502: An integrated oligonucleotide synthesis and cellular <i>in vitro</i> assay platform to support oligonucleotide-based drug discovery and development

Abstract Oligonucleotide-based therapeutics, such as small interfering RNAs (siRNAs), represent a transformative approach for gene-specific modulation in various diseases due to their capacity to regulate gene expression and protein function, with reduced toxicity compared to conventional treatments. Our oligonucleotide synthesis platform offers a comprehensive range of specialized monomers, diversified oligonucleotides, and customized solutions for oligonucleotide conjugation. To support research into oligonucleotide drugs, we have established a comprehensive in vitro assay platform that includes a series of critical evaluations such as oligonucleotide screening by transfection and RT-qPCR or western blot, oligonucleotide binding and uptake, sub-cellular localization, off-target assessment, and functional efficacy testing. For proof of concept, we employed inclisiran, one of the clinically approved siRNA therapeutics targeting PCSK9, to demonstrate our platform’s capabilities. RT-qPCR-based oligo screening assays confirmed the highly effective silencing of PCSK9, with inclisiran achieving &amp;gt;90% reduction in target mRNA, with the IC50 concentration in the single picomolar level. High levels of cellular binding, uptake and subsequent successful co-localization with endosome and lysosome within target cells were observed by means of flow cytometry or high-content imaging. The cellular binding and uptake assay verified that inclisiran maintains high affinity and selective binding of GalNAc, mediating delivery to cellular targets, while the subcellular co-localization studies demonstrated inclisiran effectively trafficking to intracellular compartments, which is essential for its gene-silencing function. Off-target assessments were conducted using a transcriptome-wide approach, which showed minimal off-target effects and confirmed specificity. Functional assessments, which included cellular LDL uptake, further validated the efficacy of inclisiran in modulating PCSK9 expression. In conclusion, we provide an integrated one-stop platform for oligonucleotide-based drug discovery and development, aiming to expedite the early-stage discovery process. Our in vitro platform allows for a thorough evaluation of oligonucleotide drugs, covering critical functional and efficacy aspects that are essential for preclinical development. Our data with inclisiran as a model therapeutic confirm the platform’s efficiency and robustness in supporting the discovery and optimization of oligonucleotide therapeutics, providing a valuable resource for advancing gene-silencing therapies. Citation Format: Di Zhan, Shuyue Wang, Qiuya Huang, Zichun Wang, Wei Xue, Yinfei Yin, Xingquan Ma. An integrated oligonucleotide synthesis and cellular in vitro assay platform to support oligonucleotide-based drug discovery and development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5502.