Exon Skipping
The exon skipping technique tries to change a Duchenne mutation into a Becker mutation. If a variation disturbs the reading frame and thus causes Duchenne dystrophy, the reading frame can be restored by artificially removing from the messenger RNA one or more exons directly in front or after the deletion, the duplication, or the exon which contains a point mutation.
Exons can be eliminated from the mRNA with antisense oligoribonucleotides, AONs. They are short RNA single-stranded structures consisting of 20 to 30 nucleotides whose sequences are constructed in such a way that they attach themselves only at the complementary sequence inside the exon to be removed or at its borders and nowhere else. Antisense means that their base sequence is in reverse order to the target sequence in the pre-mRNA. These AONs thus interfere with the splicing machinery so that the targeted exons are no longer included in the mRNA, they are skipped. The gene itself with its mutation is not altered by exon skipping, but its mRNA no longer contains the information of the skipped exon or exons. As this mRNA is shorter than normal, the dystrophin protein is also shorter, it contains fewer amino acids. If the missing amino acids are part of non-essential regions, like the central rod domains, the shorter protein can often still perform its stabilizing role of the muscle cell membrane. The result would be the change of the severe Duchenne symptoms into the much milder symptoms of Becker muscular dystrophy.
Oligonucleotides are short pieces of the two kinds of nucleic acids, DNA and RNA - oligo means few. The two strands of DNA, deoxyribonucleic acid, consist each of a chain of alternating phosphate and deoxyribose units, their backbone. Deoxyribose is a sugar molecule with five carbon atoms, and the second carbon atom has its usual oxygen atom missing. Each sugar unit carries one of the four bases on its first carbon atom. RNA, ribonucleic acid, has normal ribose units in its backbone with an oxygen on its second carbon atom. Nucleotides are the building blocks of both kinds of nucleic acids. Each nucleotide consists of one ribose, one base and one phosphate. So there are four different ribonucleotides and four different deoxyribonucleotides.
The two kinds of AONs mostly used for exon skipping are protected oligoribonucleotides so that they are not or only slowly destroyed in the muscle cells by nucleases, enzymes, that destroy nucleic acids. The Dutch scientists are using 2'-O-methyl-phosphothioates, also called methyl thioates or 2O-methyls. They have a methyl group, a carbon with three hydrogen atoms, on the oxygen of the second carbon of the ribose units, and a sulfur atom instead of one of the oxygen atoms of the phosphate groups. The morpholinos, the British and Australian researchers are using, have one of the phosphate oxygens replaced by a dimethyl amide group, a nitrogen carrying two methyl groups, and the entire ribose units are replaced by morpholino rings, six-membered rings, each consisting of four carbon atoms, one oxygen and one nitrogen atom with hydrogen atoms attached to the carbons.
(Günter Scheuerbrandt 2006)