A bioanalytical method for detection of phosphorodiamidate morpholino oligomers in cells. (65.44)

Abstract

Abstract Phosphorodiamidate morpholino oligomers (PMOs) are neutrally charged antisense agents that interfere with target gene expression by inhibition of translation or by altering splicing of pre-mRNA. PMOs have been shown to be highly specific and potent therapies after cellular uptake, yet a method to detect PMO inside the cell has not been developed. We offer in this report a novel method for the detection of cellular resident PMO using flow cytometry-fluorescence in situ hybridization (flow FISH) which takes advantage of locked nucleic acid (LNA) oligonucleotides as probes. A biotinylated LNA probe complementary to an Influenza A specific PMO (IA-PMO) was used to specifically detect and quantify intracellular IA-PMO after in vitro or in vivo administration with or without Influenza A infection. While IA-PMO was detected in non-hematopoietic and hematopoietic cells of the lung regardless of influenza virus infection, it was detected at higher levels in influenza infected hematopoietic lung cells as compared to uninfected cells. Coincident measurement of target knock down to intracellular IA-PMO concentration allowed for the calculation of an EC50. Furthermore, it was observed that an increase in IA-PMO uptake occurs in infected hematopoietic cells.