Novel Anti-Sense Oligonucleotide Delivery to Primary Lens Epithelial and Trabecular Meshwork Cell Cultures

Abstract

Purpose:: To modulate gene expression in primary lens epithelial (LEC) and trabecular meshwork cell (TMC)cultures using highly specific, nuclease resistant non-anionic morpholine-backbone anti-sense oligonucleotides (morpholinos).

Methods:: Porcine LECs and TMCs were harvested, digested, and grown to confluence. A morpholino with a fluorescent red lissamine end-modification was administered to the cultured cells with an Endo-porter delivery agent. Controls lacking either morpholino or Endo-Porter were also studied in two groups. Cells were qualitatively evaluated for distribution of intracellular fluorescence and quantitatively evaluated for amount of fluorescence by flow cytometry.

Results:: Delivery of lissamine-tagged morpholino resulted in LECs and TMCs developing a diffuse red fluorescence characteristic of ctyosolic distribution after 48 to 72 hours, compared to same-age untreated controls. After reaction optimization a significant number of LECs and TMCs successfully incorporated cytosolic morpholinos.

Conclusions:: These data indicate that morpholinos can be effectively delivered intracellulary to porcine primary cultures of lens and trabecular meshwork cells for use in anti-sense modulation of gene expression.

Keywords: gene modifiers • gene/expression • anterior segment