Novel Targeting Of COX-2 pre-mRNA Using Antisense Morpholino Oligonucleotides Directed To The 3' Acceptor And 5' Donor Splice Sites Of Exon 4: Suppression Of COX-2 Activity In Human Amnion-Derived WISH And Myometrial Cells

Increased expression of COX-2 has been implicated in the onset of both term and preterm labour. In this context both selective and non-selective COX-2 inhibitors have been used in clinical trials to determine their efficacy in delaying preterm labour. However recent evidence indicates that these tocolytics may have potential adverse fetal and maternal side effects. Consequently, the development of more specific and non-toxic agents to inhibit COX-2 needs to be considered. We have evaluated whether antisense morpholino oligonucleotides have therapeutic potential in inhibiting COX-2 by specifically targeting both the 3' and 5 ' acceptor and donor sites of exon 4 of its pre-mRNA sequence. Confocal microscopy on 'live' cells illustrated high levels of penetrance of antisense morpholino oligonucleotides using the Endo-Porter formula (Gene-Tools, LLC) with delivery efficiencies of 82% and 78% respectively in amnion-derived WISH and myometrial cells. Substantial inhibition by the morpholino oligonucleotides of COX-2 expression, induced by LPS administration, was observed at both the mRNA and protein level. Loss of enzymic activity of COX-2 was confirmed using a sensitive COX enzyme activity assay which reflects the rate of conversion of arachidonic acid to PGH2. Our results indicate that antisense morpholino oligonucleotides significantly inhibit expression and activity of this enzyme in in vitro cultures of amnion-WISH and myometrial cells. The potential thus exists that a similar approach can be mimicked in vivo to produce a highly specific and non-toxic strategy to inhibit COX-2 activity with its subsequent effects on the better management of preterm labour and other inflammatory conditions.