The influence of antisense oligonucleotide length on dystrophin exon skipping.

Antisense oligonucleotides (AOs) can be used to redirect dystrophin pre-messenger RNA (mRNA) processing, to remove selected exons from the mature dystrophin mRNA, to overcome nonsense mutations, and/or restore the reading frame. Redundancy within the dystrophin protein allows some domains to be removed without seriously compromising function. One of the challenges for splicing blockade is to design AOs that efficiently remove targeted exons across the dystrophin pre-mRNA. AOs are initially designed to anneal to the more obvious motifs implicated in the splicing process, such as acceptor or donor splice sites and in silico predicted exonic splicing enhancers. The AOs are evaluated for their ability to induce targeted exon skipping after transfection into cultured myoblasts. Although no single motif has been implicated in the consistent induction of exon skipping, the length of the AO has emerged as an important parameter in designing compounds that redirect dystrophin pre-mRNA processing. We present data from in vitro studies in murine and human cells showing that appropriately designed AOs of 25-31 nucleotides are generally more effective at inducing exon skipping than shorter counterparts. However, there appears to be an upper limit in optimal length, which may have to be established on a case-by-case basis.

Effects of prostaglandin E2 on salicylate-induced damage to the rat gastric mucosa: cytoprotection is not associated with preservation of the gastric mucosal barrier.

The effects of pretreatment with prostaglandin E2 (PGE2) on salicylate-induced gastric damage in the rat were studied with a gastric chamber model. Transmural potential difference (PD) and net potassium ion (K+) efflux were monitored as indices of gastric mucosal barrier integrity. Topical application of 20 mM salicylate for 10 min produced an abrupt drop in PD, an increase in net K+ flux, and the formation of hemorrhagic erosions covering approximately 24% of the glandular mucosa. Prior topical application of PGE2 at doses of 25, 75, and 300 micrograms/kg significantly reduced the extent of hemorrhagic erosions. However, PG pretreatment did not produce a reduction in the effects of topical salicylate on either PD or net K+ efflux. Rather, the drop in PD was initially accelerated and the net K+ efflux was increased by PGE2 pretreatment. Subsequently, in PGE2-pretreated mucosae, both parameters showed more rapid recovery toward control values. These results suggest that the mechanism of "cytoprotection" by PGE2 against salicylate-induced gastric damage is not a consequence of preservation of the gastric mucosal barrier.

Correlations between changes in indicators of gastric mucosal barrier integrity at time of exposure to "barrier breakers" and extent of hemorrhagic erosions one hour later.

We have examined the effects of seven different "barrier breakers" (including ethanol, aspirin, salicylic acid, isobutyric acid, Na taurocholate, thermal injury, and hyperosmotic glucose) on chambered gastric mucosae of rats in an attempt to identify variations in accepted indicators of mucosal barrier integrity which would accurately predict the extent of subsequent hemorrhagic erosion. When results from all experimental groups were considered, only the initial decrease in transmucosal potential difference (PD) showed significant correlation with final damage (lesion area). When the results were analyzed as separate subgroups, significant correlations were also found between net K+ efflux during the first 10 min after luminal infusion and final lesion area. Only in the subgroup containing ethanol, salicylates, and thermal injury was there a correlation between net loss of luminal H+ (back-diffusion) and lesion area. These results are considered in terms of their implications for the ulcerogenic actions of each group of agents.