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Exon Skipping

When you tell someone that your child has Duchenne Muscular Dystrophy, more often than not you get this blank look from them while they try to gauge your demeanor to figure out just how serious it is. Most people have at least heard of the disease, but the impact of DMD isn't something the average person tracks until it affects them, and it's difficult to reconcile that someone as happy and full of energy as Talen could be afflicted with a terminal wasting disease.

Once the severity sinks in, one of the first questions people ask is, "Can they treat it?" Currently the answer is, "Not really." There are a number of treatment options under investigation, and I have been attempting to catalog avenues of research at Toomee.org. I have not had time to work on it recently, but I'm committed to comprehensively logging all of the research that is publicly available.

However, there are a couple of items that I think are very close to being accessible which bear addressing specifically. I'll dive into details after the jump, but the two main avenues of investigation that we are excited about right now are exon skipping and utrophin upregulation. We'll dive about an inch deep into exon skipping here and talk about utrophin in another post.

Exon skipping (and genetic biology in general) can seem like a very daunting topic, and I don't want to seem dismissive of its complexity, but it is possible to gain a basic understanding of exon skipping without diving too deep into genetics. I think, at a minimum, you need to understand the following:

  • The primary relevant function of a gene is to encode/create amino acids which are assembled into proteins.
  • Encoding begins with one of four nucleotides (five if you count Uracil, which replaces Thymine in RNA but that's not really important for this discussion).
  • Exons are sections of the genetic sequence used to encode protein (not technically accurate but we're not going to get into the details of translation and transcription here so it's good enough for a short discussion)
  • The four base nucleotides are assembled in triplets to encode amino acids in sequence.
  • These triplets have to be assembled in specific order or the result will be something other than the expected amino acid (and the protein chain cannot be completed)
  • DMD is commonly caused (but not always, how is that for non-specificity?)by deletions known as frameshift mutations, meaning that the missing bases cause the remaining base sequences to combine into useless combinations.
  • Exon skipping turns a frameshift mutation into an in-frame mutation by hiding chunks of genetic code from the translation/transcription process, which results in base pairs that are once again sequenced in triplets to produce actual amino acid chains that can be used to produce proteins (dystrophin for example).

There is a slightly more in depth article that is still meaningful to non-geneticists on the Leiden University site: [See more info here](http://www.humgen.nl/lab-aartsma-rus/index%20for%20parents.html)

Leiden University, which I had never heard of before Talen's diagnosis, is one of the earliest research institutions to investigate exon skipping through the use of morpholino oligomers. Bit of trivia here: morpholino oligomers use as their structural base an organic compound (morpholine rings) that are used as a fossil fuel additive. Chemistry majors are probably rolling their eyes here, but I still think it's fascinating that the same compound used in gasoline can be used to treat genetic mutations.

So it's worth noting that exon skipping isn't some magic bullet. The resulting protein that is encoded from the truncated sequence is still missing information, and you have to assume that all of the information is important to some degree. What makes exon skipping so attractive for a large group of DMD patients is that a large number of mutations occur in the middle of the sequence, and this part of the protein is largely repetition of the same sequence. Losing part of the sequence in the middle is usually tolerated better than losing pieces at either end. Since dystrophin's primary function is to anchor cell membranes, the shape of either end is important as it affects the protein's ability to attach to either layer. This is important to note, because if the mutation occurs early or late in the sequence, exon skipping probably won't be of any benefit (doesn't matter if you are in frame when you can't attach to hold the cells together).

If you've stuck with me this far, you might already be able to guess the biggest issue with exon skipping. In a large gene (2.6ish million bases in the dystrophin gene) there are a number of places that deletions can occur. Kids with DMD have deletions throughout the gene, with a substantial number clustered in the middle region of the gene. Exon skipping relies on masking or hiding the genetic information immediately prior to or after the deletion to restore the reading frame, so the targets for masking are myriad. Each specific mutation requires a specific compound to restore the reading frame, unless you mask a larger section of the gene than necessary.

In Talen's case, the deletion occurs from exons 48-54. Leiden University has given us a "Exon Skipping for Dummies" graphic that will let us determine which exons can be skipped to produce the desired results.

If you are able to read the whole chart (I think my Ipad isn't cut out for updating blog posts), you will notice that each numbered exon is given a shape that fits with the exon preceding and following. Look at exon 58 for example. It fits right into the groove of exon 59 and exon 57. If you deleted exon 59, exons 58 and 60 don't fit together. However, if you were to hide exon 58, exon 57 will fit nicely into exon 60. This is the super simple version of how exon skipping works.

So look at 48-54 and pretend they were missing. 47 and 55 do not fit together, but 47 and 56 DO, so masking exon 55 would restore the reading frame for someone with Talen's mutation.

The number of kids who would benefit from any specific exon skipping compound is relatively small, which makes research and development less than enticing for pharmaceutical industries. The biggest target, exon 51, addresses somewhere around 13% of the DMD population, which is tens of thousands. Not a big money maker, so a lot of parents are being left out in the cold.

Luckily, Prosensa has in its preclinical portfolio a compound that will mask exon 55, and has recently received orphan status (preferential treatment) for all of its preclinical drugs, so we will likely see this go into clinical trials later this year if we are lucky.

This is without a doubt the most promising and likely available treatment option that is looming around the corner. The preliminary data from the exon 51 skipping trials seemed to show little improvement, but after several additional months researchers started to see statistically significant gains in children receiving treatment, versus declines in children on placebo.

This post turned into something way longer than I expected, so I won't get too deep into the current status or the trial results from exon 51 skipping, but I'll come back to it in another post. Suffice to say that we are excited and watching the interwebs for any news on Prosensa and exon 55 skipping.