Introns, exons, and stop codons: how antisense oligonucleotides can fix frameshift mutations

DMD is the largest gene in nature, covering roughly 2.4 mega bases of the X chromosome. It encodes the dystrophin protein, a component of the protein complex that connects the cytoskeleton to the extra-cellular matrix.

DMD is a very complex gene. Its RNA transcripts are differentially spliced, which means that the gene produces different transcripts, encoding a large set of protein isoforms. A refresher: every gene is composed of coding parts, called exons, interspersed with non-coding bits, called introns. When the gene is transcribed into RNA, a process called RNA splicing, the introns are removed and the exons (grr… my auto-correct keeps turning all my "exons" into "eons"!) reassembled to form the RNA transcript that will be used to form proteins. Some proteins, like dystrophin, have different isoforms (some specific to different cell types), and those are obtained through different splicing forms of the RNA, originated by maintaining a different number of exons in the final transcript.

This video is a good illustration of RNA splicing:

All this to give you an idea of how complex this gene is. So, when it carries a mutation, things get very complicated, and the consequences devastating. Mutations in the DMD gene are responsible for several forms of muscular dystrophy (MD), and because the gene is on the X chromosomes, the prevalence is usually higher in boys than girls. (This is because girls carry two X chromosomes, hence if one allele only is mutated, the other will compensate.)

The most common mutations causing muscular dystrophy cause the transcription process to stop too early, producing incomplete, and therefore non-functional, RNA transcripts. I discussed reading frames in this post: in layman terms, the reading frame of a gene is how you split the bases in triplets so that each triplet codes one amino acid (the building blocks of proteins). Mutations that cause a shift in the reading frame basically disrupt the translation into amino acid, often resulting in the early termination of the transcription process (when the frameshift causes the random appearance of an early stop codon). When not enough functional dystrophin is produced, individuals experience a significant loss in muscle function and muscle degeneration.

How to counteract the action of frameshifting mutations?

One way is to use antisense oligonucleotides, buts of RNA that bind to a splicing site on the pre-mRNA causing the deleterious exons to be skipped and thus restoring the "functional frame." What does this mean? Remember, RNA is one-stranded. From DNA to RNA there are several steps: pre-messenger RNA and messenger RNA, or mRNA. The deleterious mutations are on the gene and they cause a misread when going from DNA to pre-mRNA. Now the mutations are on the pre-mRNA. Suppose you can devise a "bandage" that literally covers the bit of bases causing the framshift. If the bandage works, the bit won't be read when the pre-mRNA is turned into mRNA thus effectively canceling the frameshift and restoring the original RNA transcript. These "bandages" are bits of antisense RNA specifically made to bind to the "bad" parts of pre-mRNA. I covered this kind of therapy in an earlier post on gene therapy.

Does it work? So far, enough to give hope.

Goemans et al. [1] recruited 12 patients with Duchenne's muscular distrophy. Over the course of 12 weeks, the patients received weekly, dose-escalating

"weekly abdominal subcutaneous injections of PRO051 (from 0.5 to 10 mg per kilogram of body weight, with 3 patients receiving each dose) for 5 weeks. The specific increases in dose were determined after analysis of safety and dystrophin levels in muscle-biopsy specimens."

The lowest dose of 0.5 mg per kilogram showed no effect on RNA or protein expression. Exon-skipping RNA was instead observed in the higher-dose patients. Muscle biopsies were sampled at the end of the high-dose period and new dystrophin expression was observed starting from week 2, with increased signal as time and dose progressed. By the end of the twelve weeks the average distance walked in six minutes across all patients had increased by 35 meters, with some patients able to walk 65 meters farther than at baseline. Patients were also tested 2 and 7 weeks after the treatment, and most still showed similar dystrophin expression levels as right after the treatment.

Though a lot still needs to be done in order to defeat this disease, these results certainly set a much needed step forward.

[1] Goemans, N., Tulinius, M., van den Akker, J., Burm, B., Ekhart, P., Heuvelmans, N., Holling, T., Janson, A., Platenburg, G., Sipkens, J., Sitsen, J., Aartsma-Rus, A., van Ommen, G., Buyse, G., Darin, N., Verschuuren, J., Campion, G., de Kimpe, S., & van Deutekom, J. (2011). Systemic Administration of PRO051 in Duchenne's Muscular Dystrophy New England Journal of Medicine, 364 (16), 1513-1522 DOI: 10.1056/NEJMoa1011367

This picture was a "Rule of Thirds" exercise: find an interesting background, and have the main subject of your photo cover one third of the picture only, instead of positioning it in the middle. You can see how it immediately makes both subject and background more interesting. Focal length 26mm, shutter speed 1/25, ISO speed 100, F-stop 7.1