About exon skipping
What is exon skipping and how does it work?
In order to explain the concept of exon skipping, it is first necessary to explain how genes work and how mutations in the dystrophin gene can cause both Duchenne and Becker muscular dystrophy.
What are genes?
DNA is an extremely long molecule which contains the instructions to create and maintain our bodies. A gene is a section of DNA that contains the instructions for the production of one specific protein. Proteins are essential parts of cells and play a role in every process occurring within the cell, as well as having structural or mechanical functions which help maintain the cells' shape. It is estimated that we have about 30,000 different genes.
What are exons?
Genes are divided into sections called exons and introns. Exons are the sections of DNA that code for the protein and they are interspersed with introns which are also sometimes called 'junk DNA'. The introns are cut out and discarded in the process of protein production, to leave just the exons. The dystrophin gene is our largest gene- it has 79 exons which are joined together like the pieces of a puzzle.
Seen above is the diagram of the 79 exons of the dystrophin gene which fit together like the pieces of a jigsaw puzzle.
What happens in Becker muscular dystrophy?
Let's zoom in on exons 48 to 54 to look at this a bit more closely:
In Becker muscular dystrophy an exon is deleted, for example exon number 53 in the diagram:
Although a part of the gene is missing, exon 52 can join up with exon 54, and the puzzle can be completed to the end of the gene:
What impact does a Becker mutation have on the dystrophin protein?
The dystrophin protein normally sits in the membrane that surrounds muscle fibres like a skin, and protects the membrane from damage during muscle contraction. Without dystrophin the muscle fibre membranes become damaged and eventually the muscle fibres die.
Dystrophin is a very large protein with a section in the middle consisting of lots of repeated segments (in green below) and it is known that the protein can still work to some extent if some of these repeated segments are missing. Individuals with Becker muscular dystrophy have some of these repeated segments missing and have relatively mild symptoms- often being able to still walk into their 40s and 50s.
Diagram of the dystrophin protein
A man has even been known to be still walking at 61 years of age, despite having a deletion of 46% of the dystrophin gene!
Illustration of the dystrophin protein in Becker muscular dystrophy
What happens in Duchenne muscular dystrophy?
In Duchenne muscular dystrophy an exon, or exons are deleted which interfere with the rest of the gene being pieced together. In our example, exon 50 illustrates this:
Exon 49 can not join up with exon 51, which prevents the rest of the exons being assembled. For the dystrophin protein to work it must have both ends of the protein. Therefore, this mutation results in a completely non-functional dystrophin protein and the severe symptoms of Duchenne muscular dystrophy.
How can exon skipping help?
As the name suggests, the principle of exon skipping is to encourage the cellular machinery to 'skip over' an exon. Small pieces of DNA called antisense oligonucleotides (AOs) or 'molecular patches' are used to mask the exon that you want to skip, so that it is ignored during protein production. In our example, if we use a 'molecular patch' designed to mask exon 51:
Exon 49 can now join up to exon 52 and continue to make the rest of the protein, with exons 50 and 51 missing in the middle:
Therefore, exon skipping may be able to reduce the symptoms of Duchenne muscular dystrophy, to those more like Becker muscular dystrophy.
Does this really work?
So far scientists have shown this technique to be effective in a mouse model of Duchenne muscular dystrophy (the mdx mouse) and in human Duchenne muscular dystrophy muscle cells grown in the laboratory.
A phase 1 clinical trial involving nine boys with Duchenne muscular dystrophy was completed in December 2008. In this trial a 'molecular patch' (called AVI-4658) was injected into a muscle in the foot. This resulted in dystrophin production in this muscle and no serious side-effects were observed. This trial was intended to prove the principle of the technique and wasn't expected to show any improvement in symptoms since only one small muscle was injected.
Preliminary results from the next phase of the trial were released in June 2010. In this trial the molecular patch was injected into the blood stream of boys with Duchenne. At the higher doses dystrophin production was seen in muscle biopsies taken at the end of the trial. Read more about these preliminary results.
Is there a clinical trial?
Preliminary results from the UK exon skipping trial which delivered the molecular patch to the whole body via the bloodstream were encouraging but we are still waiting for news of the full results. Indications are that although dystrophin production was seen in the muscles of the boys receiving the higher doses, the response was highly variable from boy to boy, with some only producing very small amounts of dystrophin. AVI Biopharma now plans to test higher doses to try to gain a consistently strong response to the molecular patch. They are planning to conduct this trial in the US.
The Dutch pharmaceutical company Prosensa is testing a slightly different chemical formulation of molecular patch in Europe and in partnership with GSK in the US.
The challenge with all of these trials is to produce enough dystrophin in as many muscles as possible (including the heart) to prolong and improve the quality of life for boys with Duchenne muscular dystrophy.
Will it work for everyone with Duchenne muscular dystrophy?
It is thought that skipping one or two exons would be able to treat around 83% of the genetic errors causing Duchenne muscular dystrophy.
Will the same 'molecular patch' work for everyone?
No, the dystrophin gene is very large and the genetic errors associated with Duchenne muscular dystrophy occur in different places along this gene. There are however some common areas for mutations and initially 'molecular patches' will be made for these. The clinical trials are starting with exon 51 which would be applicable for around 13% of boys. Once the technology has been shown to be effective for a particular error it will be possible to design other 'patches'.
Are 'molecular patches' a cure?
Scientists hope that this type of therapy will halt or even reverse the symptoms of Duchenne muscular dystrophy so that the symptoms are more like those of boys with Becker muscular dystrophy. It will not be a cure because if proven to be effective, this treatment would need to be repeated regularly- how often will become apparent during clinical trials.