Standard ASO design is based on the sequence complementarity of the oligo to its target. However, the degree of target knockdown that ASOs can achieve varies strongly between different ASOs having full complementarity to the target. To determine which factors affect the ASOs’ activity, Secarna has used a novel approach: the Company has designed and screened 51 multi-specific ASOs with a common target (IDO1, a gene involved in tryptophan metabolism), and varying numbers of diverse other targets (up to approx. 2,500). In a first step, Secarna investigated the ASO’s effectivity against IDO1. Subsequently, the impact of nine selected ASOs on the overall gene expression profile was further investigated by RNA-sequencing. The resulting dataset formed the basis for the examination of the roles of different factors that may influence ASO activity, allowing Secarna to base their findings on an unpreceded database of thousands of genes.