Abstract
Antisense oligonucleotides (ASOs) are short, single-stranded nucleic acid molecules that alter gene expression. However, their transport into appropriate cellular compartments is a limiting factor in their potency. Here, we synthesized splice-switching oligonucleotides (SSOs) previously developed to treat the rare disease erythropoietic protoporphyria. Using chemical ligation-quantitative polymerase chain reaction (CL-qPCR), we quantified the SSOs in cells and subcellular compartments following free uptake. To drive nuclear localization, we covalently conjugated nuclear localization signal (NLS) peptides to a lead 2'-O-methoxyethyl phosphorothioate SSO using thiol-maleimide chemistry. The conjugates and parent SSO displayed similar RNA target-binding affinities. CL-qPCR quantification of the conjugates in cells and subcellular compartments following free uptake revealed one conjugate with better nuclear accumulation relative to the parent SSO. However, compared to the parent SSO, which altered the splicing of the target pre-mRNA, the conjugates were inactive at splice correction under free uptake conditions in vitro. Splice-switching activity could be conferred on the conjugates by delivering them into cells via cationic lipid-mediated transfection or by treating the cells into which the conjugates had been freely taken up with chloroquine, an endosome-disrupting agent. Our results identify the major barrier to the activity of the peptide-oligonucleotide conjugates as endosomal entrapment.