Effects of cocktail combinations of antisense oligonucleotides on potency

Abstract Background: The co-administration of several therapeutic oligonucleotides targeting the same transcript is a beneficial approach. It broadens the target sites for diseases associated with various mutations or splice variants. However, little is known how a combination of antisense oligonucleotides (ASOs), which is one of the major modalities of therapeutic oligonucleotides, affects the potency. In this study, we aimed to elucidate the cocktail-effects of ASOs and the relationship between the target sites and potency of different combinations. Method and Results: We designed 113 ASOs targeting human superoxide dismutase 1 pre-mRNA and found 13 ASOs that had comparable silencing activity in vitro. An analysis of cocktail-effects on the silencing potency of 38 pairs of two ASOs on HeLa cells revealed that 30 pairs had comparable potency to that of two ASOs; on the other hand, eight pairs had reduced potency, indicating a negative impact on the activity. A reduced potency was seen in pairs targeting the same intron, exon-intron combination, or two different introns. The sequence distance of target sites was not the major determinant factor of cocktail-effects. In addition, a cocktail of three ASOs preserving the potency could be designed by avoiding two-ASO pairs, which had a reduced potency. Conclusions: This study revealed that more than half of the combinations retain their potency by paring two ASOs; in contrast, some pairs had a reduced potency. This could not be predicted only by the distance between the target sites.