Deletions and point mutations in the gene encoding the cytoskeletal protein dystrophin and its isoforms cause either the severe progressive myopathy Duchenne muscular dystrophy (DMD) or the milder Becker muscular dystrophy (BMD), largely depending on whether the reading frame is lost or maintained respectively. Frameshift mutations tend to result in a lack of dystrophin at the sarcolemma, destabilization of the membrane and degeneration of skeletal muscle. The mdx mouse is a valuable animal model of DMD as it bears a nonsense point mutation in exon 23 of the murine DMD gene leading to an absence of dystrophin expression in the muscle sarcolemma and muscular dystrophy. This report represents a novel approach to correct dystrophin deficiency at the post-transcriptional level by transfection of muscle cells with antisense RNA. Essentially, 2'- O -methyl oligoribonucleotides (2'OMeRNA) were delivered to the nuclei of primary mdx myoblasts in culture. Dystrophin expression was observed in the sarcolemma of transfected mdx myotubes after transfection by an oligonucleotide complementary to the 3' splice site of murine dystrophin intron 22. Direct sequencing of RT-PCR products from these cells revealed precise splicing of exon 22 to exon 30, skipping the mutant exon and creating a novel in-frame dystrophin transcript. As patients with comparable in-frame internal deletions show relatively mild myopathic symptoms, this may in the future offer a therapeutic approach for DMD, as well as for other inherited disorders.

The 5' cap structure of mRNA is a N7 methylated guanosine residue that is linked by a 5'-5' triphosphate linkage to the 5'-terminus of cellular and viral RNAs synthesized by RNA polymerase II. This unique structure facilitates several processes of mRNA metabolism, including splicing, nucleocytoplasmic transport,initiation of translation, and degradation. Previous research has demonstrated that the lanthanide macrocycle complex, Eu(THED)3+, effectively cleaves the 5' cap structure of mRNA in solution by nucleophilic attack of the triphosphate linkage via the metal-activated hydroxyethyl group of the THED ligand. This report shows that attachment of a Eu(THED)3+analog to the 3'-terminus of an antisense oligonucleotide, which targets the 5'-terminus of the intercellular adhesion molecule 1 mRNA, potentiates the inhibitory activity of the antisense oligonucleotide in cytokine-treatedendothelial cells.

Single-stranded oligodeoxynucleotides (ODNs) were tested for their ability to stimulate NK cells isolated from murine spleens to lyse target cells. Various sequences were evaluated, some of which have been shown previously to exhibit pharmacologic activity in murine model systems. It was confirmed that the CpG motif was stimulatory only in specific sequence contexts, and we found that phosphorothioate backbones were, in general, less stimulatory than phosphodiester backbones. In addition, this stimulation could be reduced by methylating the cytosine of the CpG and eliminated by modifying all of the cytosines contained in an ODN with methyl, bromo, or iodo modifications to the 5 position of the cytosine ring. These results were compared with the ability of a subset of these ODN sequences to stimulate B cell proliferation in vitro. In this comparison, phosphorothioate backbones were found to be required, and the context of the CpG motif was found to be less critical for activation. Finally, one of the most potent ODNs was shown to activate NK and B lymphocytes when administered in vivo.