Some ASOs that bind in the coding region of mRNAs and induce RNase H1 cleavage can cause increases in the pre-mRNAs that may blunt total activity.

Antisense oligonucleotide (ASO) drugs that trigger RNase H1 cleavage of target RNAs have been developed to treat various diseases. Basic pharmacological principles suggest that the development of tolerance is a common response to pharmacological interventions. In this manuscript, for the first time we report a molecular mechanism of tolerance that occurs with some ASOs. Two observations stimulated our interest: some RNA targets are difficult to reduce with RNase H1 activating ASOs and some ASOs display a shorter duration of activity than the prolonged target reduction typically observed. We found that certain ASOs targeting the coding region of some mRNAs that initially reduce target mRNAs can surprisingly increase the levels of the corresponding pre-mRNAs. The increase in pre-mRNA is delayed and due to enhanced transcription and likely also slower processing. This process requires that the ASOs bind in the coding region and reduce the target mRNA by RNase H1 while the mRNA resides in the ribosomes. The pre-mRNA increase is dependent on UPF3A and independent of the NMD pathway or the XRN1-CNOT pathway. The response is consistent in multiple cell lines and independent of the methods used to introduce ASOs into cells.