To scramble or not: Steric blocking 2'-MOE/PS control antisense oligonucleotides across cellular models

Antisense oligonucleotides (ASOs) are emerging as a promising therapeutic approach for many disorders. Currently, fourteen ASOs have been FDA/EMA-approved, with many more in preclinical development. An important requirement for preclinical studies is the inclusion of negative controls to properly assess the efficacy of candidate ASOs. As commonly used scrambled or sense ASOs may elicit unwanted off-target effects that could influence downstream data analysis, there is a high need for the inclusion of additional negative control ASOs (cASOs) that are well-characterized and well-tolerated. In this study, we designed and evaluated generic cASOs across seven different cellular models. Characterization at the RNA and protein level revealed potential ASO toxicity mechanisms as well as species-specific and cell-type-specific effects. In total, three steric blocking 2'-MOE/PS cASOs with a distinct GC content were identified that exert limited off-target effects across all cellular models. To conclude, we report a set of cASOs that are potentially applicable in a broad variety of experiments using ASOs with similar physicochemical properties and across different cellular models.