Optimizing Delivery of Novel Anti-Sense Oligonucleotides to Trabecular Meshwork Cells

Purpose: To assess and refine the delivery of highly specific, nuclease resistant non-anionic morpholine-backbone anti-sense oligonucleotides (morpholinos) to porcine trabecular meshwork (TM) cells for modulating gene expression. Methods: Porcine TM cells were harvested, digested, and grown to confluence. A morpholino with a fluorescent red lissamine end-modification was administered to the cultured cells in two groups, with and without an Endo-porter delivery agent. Controls lacking either morpholino or Endo-Porter were also studied in two groups. Trabecular meshwork cells were qualitatively evaluated for distribution of intracellular fluorescence and quantitatively evaluated for amount of fluorescence by flow cytometry. Results: Sole delivery of lissamine-tagged morpholino resulted in TM cells developing a diffuse red fluorescence characteristic of ctyosolic distribution after 72 hours, compared to same-age untreated controls. Cellular toxicity was observed with the delivery of Endo-porter and varied depending on the concentration of morpholino and Endo-porter. After reaction optimization a significant number of TM cells successfully incorporated cytosolic morpholinos. Conclusions: These data indicate that morpholinos can be effectively delivered intracellularly to porcine TM cells for use in anti-sense modulation of gene expression.