Antisense oligonucleotide-based switches for conditional translation of synthetic mRNA

Synthetic messenger RNA (mRNA) therapeutics requires precise control over their translation to minimize off-target effects. We introduce an antisense oligonucleotide (ASO)-based switch concept that regulates synthetic mRNA translation based on the presence of a complementary trigger RNA (trigRNA). We evaluate variations in ASO hybridization length, position, and chemistry in rabbit reticulocyte lysate (RRL) assays and HEK293T cells. In addition, we explore multiple ASO delivery strategies, including lipid and dendrimer conjugation. Our experiments demonstrate that increased ASO hybridization length and concentration enhance suppression while also improving the OFF/ON dynamic range in the presence of trigRNA. Cell experiments revealed that cholesterol or dendrimer-conjugated phosphorodiamidate morpholino oligonucleotides (PMOs) improved uptake leading to increased translational suppression which facilitated moderate trigRNA-dependent activation. Alternatively, PMOs that exhibit higher complementarity for the trigRNA than for the mRNA reached a dynamic range of 86.4 in RRL and 1.79 in HEK cells. We conclude that the presented ASO-based switch concept provides a novel mRNA translation control strategy, which does not rely on expressing exogenous proteins. While RRL assays demonstrate robust switching, intracellular efficacy is currently constrained by ASO delivery and intracellular dilution. Future optimization of ASO stability and maintaining intracellular suppression will be critical for therapeutic applications.