In Vitro Exploration of Antisense and Splice-Switching Oligonucleotide Multimers Using an Engineered Peptide Scaffold.

Antisense oligonucleotides (ASOs) are potent therapeutics that enable precise RNA modulation. However, efficient delivery to tissues and cell types remains a major challenge. Multimerization, linking multiple ASOs to form large constructs, represents a promising strategy to explore delivery and pharmacokinetics modulation. In this study, we utilized an engineered peptide scaffold to synthesize and evaluate multimeric ASOs, with up to four ASOs connected, with or without a delivery ligand (palmitic acid, bicycle-TfR1, or GalNAc). We demonstrate that ASO multimerization does not impair target annealing, ligand binding, or silencing activity but displays endosomal entrapment in vitro, requiring nanoparticle delivery. Using a luciferase reporter assay, we further assessed the strategy's potential for splice-switching oligonucleotides by designing chimeric constructs capable of targeting two splicing sites at once. Our findings establish the synthetic feasibility of the multimers, identify endosomal entrapment as a key in vitro limitation, and outline considerations for future in vivo evaluation.