Intracellular delivery strategies for antisense phosphorodiamidate morpholino oligomers

Antisense oligonucleotides inhibit gene expression by interfering with transcription, translation, or splicing. They show great potential as gene-specific, nontoxic therapy for a wide variety of diseases. They are also powerful tools to study gene function as well as for validation of therapeutic targets. Even with compelling evidence of activity in vivo, the majority of cell types in culture require technologies capable of efficiently delivering antisense oligonucleotides into the cytosolic/nuclear compartment of the cells in culture. Phosphorodiamidate morpholino oligomers (PMO) are a new generation of antisense oligomers with high specificity and efficacy. They inhibit translation of targeted mRNA by steric blockade. Different methods were evaluated for efficient delivery of PMO into the cells in culture. Efficacy was compared using the PMO targeted to the 5'-untranslated region (5'-UTR) of alpha-globin-luciferase reporter fusion gene mRNA. A functional assay based on the lunciferase reporter system was used to measure efficacy. The fluorescence-activated cell sorting (FACS) method was used for quantitative determination of PMO uptake into the cells. Physical methods, such as scrape-loading, syringe-loading, and osmotic-loading, provided efficient transfer of PMO into the cells, which resulted in higher efficacy. These procedures caused minimal damage to the cells. Cell permeabilization with streptolysin O did not improve the cell uptake of PMO. Complexation with cationic lipids, Lipofectin and Lipofectamine (GIBCO-BRL, Gaithersburg, MD) also failed to enhance the uptake of PMO. We conclude that physical methods are optimal for the delivery of neutrally charged PMO into cells in culture. Further, these methods do not leave residual material that may interfere with the interpretation of targeted gene function.