Duchenne muscular dystrophy - 'molecular patches' as a therapy - Application to the Department of Health

Duchenne muscular dystrophy - 'molecular patches' as a therapy. A Department of Health application to move research towards the clinic.

Written by the Research Department at the Muscular Dystrophy Campaign.

This question and answer document is aimed at explaining the ‘molecular patch’ technique as a therapy for Duchenne muscular dystrophy and a recent application to the Department of Health (DoH) to fund this innovative research.

Contents:


What causes Duchenne muscular dystrophy and the milder Becker muscular dystrophy form?

Both are caused by genetic errors in the dystrophin gene, which affect the production of an essential muscle protein called dystrophin. Without functional dystrophin protein, muscle cells begin to weaken and eventually die.

What is the difference between Duchenne muscular dystrophy and Becker muscular dystrophy?

In Duchenne muscular dystrophy, there is a total or nearly total absence of functional dystrophin protein. In Becker muscular dystrophy, a shortened dystrophin protein, which is partially functional, is produced which means the disease is less severe.

How are proteins made?

A basic understanding of this is necessary to understand the therapeutic approach discussed later.

The genes we inherit from our parents contain genetic code called deoxyribonucleic acid (DNA). This can be likened to a string made up of four different letters (ATGC) arranged in a particular order that is unique for each gene. This genetic code is read to form proteins. These letters can be compared to our alphabet as different combinations of letters can create thousands of different words.

To make a protein there are three essential steps:

i) a copy of the coded DNA is made (called transcription),
ii) this copy is edited to remove any non-essential information (called splicing),
iii) the remaining code is read by the cell’s machinery in groups of three letters (called translation) to form the protein. These groups of three’s make up the reading frame.

These steps can be explained using an example of letters of the alphabet:


THEWTOMMANATIANDPIQHIS URDIDOGITRANOVINFOR TRIATHEDOOPIBUS


As you can see, this set of letters (representing the DNA) cannot be read because there are extra letters (known as junk DNA or introns, indicated in bold) which need to be removed before the words can make sense. This process of removing the junk is called splicing.

The DNA is copied into RNA and then edited so that only the code essential for making the protein is left:


THEMANANDHISDOGRANFORTHEBUS

If one looks closely and reads this code in groups of three you will see that it reads:


THE MAN AND HIS DOG RAN FOR THE BUS

If both Duchenne muscular dystrophy and Becker muscular dystrophy are caused by errors in the dystrophin gene, why is one more severe than the other?

In Duchenne muscular dystrophy the common errors either stop the production of protein because of a fault in the beginning part of the code, or the error changes the code so that it no longer makes sense. In the latter example we say that the error affects the “reading frame”.

If we visualise this by imagining the error to be the removal of the letters ND in the example above – (remember the code is read in groups of three): this leaves us with coding which makes no sense and cannot produce any functional protein:

                       ND-Part of the code that is missing
THE MAN AHI SDO GRA NFO RTH EBU S


In this example only two letters are missing yet it has a catastrophic affect because the reading frame is altered.


Duplications (adding letters) in the dystrophin gene can also affect the reading frame and in these instances there is also potential to use ‘molecular patches’ to restore the reading frame.

What happens in Becker muscular dystrophy?

In Becker muscular dystrophy, the loss of genetic code involves groups of three’s, and the reading frame  is not affected. The result is a shorter piece of genetic code, which still makes some sense and produces a shorter but partially functional dystrophin protein:

For example the letters     ANDHISDOG are missing.

This leaves us with:  THE MAN RAN FOR THE BUS

This sentence is not complete but at least it makes sense and a partially functional protein is produced. You can see that the deletion in this example is larger than the example above causing Duchenne muscular dystrophy but because it is a multiple of three it does not disrupt the reading frame. There is still some sense, which means that some protein can be produced.



Can we turn a Duchenne muscular dystrophy type mutation into a Becker muscular dystrophy type mutation?

Yes, this is the objective of using ‘molecular patches’.

What is the ‘molecular patch’ or ‘exon skipping’ technique?

It involves making a very small piece of genetic material (‘molecular patch’), which once inside a muscle cell, will bind to its matching sequence of genetic code. This ‘patch’ is designed so that it binds a region surrounding the genetic error. When editing takes place to remove the non-essential regions of code, the area covered by the patch is not included in the final sequence which goes on to produce the protein. In this way the reading frame is altered so that it becomes readable. This may be clearer using the following example:

Consider the mutation we used above where the letters ND are missing. If a ‘patch’ is made to bind to the letters AHISDOG, and delivered to muscle cells, it will bind to its matching genetic sequence. During editing the AHISDOG will not be included and we are left with:


THE MAN RAN FOR THE BUS


The effect of this is to turn a sentence, which could not be read, into one which can.  With the dystrophin gene, this is the difference between no protein being produced and a Becker muscular dystrophy-like protein being produced.



Does this really work?

So far scientists have shown this technique to have therapeutic effect in a mouse model of Duchenne muscular dystrophy (the mdx mouse) and in human Duchenne muscular dystrophy muscle cells grown in the laboratory. It has not yet been tested in living human beings so it is therefore very important that we perform initial safety trials.

Do scientists understand how this works in the body?

Not fully. Scientists need to study the editing process further so that we can optimise the use of these ‘patches’ to make them work more efficiently and have maximum therapeutic benefit.

Will it work for everyone with Duchenne muscular dystrophy?

No, but it is thought that around 60% of the genetic errors associated with Duchenne muscular dystrophy could be treated with the use of ‘molecular patches’.

What other options are there if ‘molecular patches’ are not suitable for a particular type of genetic error?

This technique is just one of many identified by scientists as having therapeutic potential. Researchers world-wide are investigating many different approaches which may result in other therapies. These include techniques such as transferring a working copy of the dystrophin gene and drug treatments.

How do you determine whether ‘molecular patches’ will be helpful as a treatment for a particular type of mutation?

There are specialised tests, which enable scientists to determine the exact nature of your genetic error. In many cases the doctors already know this information, especially if you have been diagnosed in recent years.

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 to prove that the technique works. It is thought that several different ‘patches’ will be required to cover the spectrum of genetic errors. Once the technology has been shown to be effective for a particular error it will be possible to design other ‘patches’.

Will ‘molecular patches’ help the more severe forms of Becker muscular dystrophy?

The effectiveness of ‘molecular patches’ is not dependant on the condition, in fact it has been shown to be therapeutic in other related conditions. The key issue is whether altering the way in which the genetic code is read has a therapeutic effect. There may be certain instances where ‘molecular patches’ might be helpful in severe forms of Becker muscular dystrophy; however most individuals with Becker muscular dystrophy will probably not benefit from this approach.

I have heard about an application to the Department of Health, what does this involve?

The three charities involved in Duchenne muscular dystrophy in the UK are the Muscular Dystrophy Campaign, Parent Project UK (PPUK) and Duchenne Family Support Group (DFSG). Together they have been lobbying in Government for extra funding towards research for Duchenne muscular dystrophy and this resulted in a meeting with the Secretary of Health, Lord Warner. Around the same time the Government announced a special allocation of £3 million for gene therapy related to single gene disorders. It was suggested at this meeting that we apply for part of this money and a Consortium of eminent scientists have worked together to put in this application. The aim of this project is to:

• optimise the structure of these ‘molecular patches’;
• improve delivery techniques to try and reach as much muscle as possible;
• test these ‘molecular patches’ in animal models and cultured human Duchenne muscular dystrophy cells;
• perform the first human safety trial to test the safety of the ‘molecular patch’ and see if functional dystrophin is produced;

Who will participate in the initial safety trial?

Participants will be selected using specific inclusion criteria. These will include age (probably over the age of 14), the type of genetic error causing Duchenne muscular dystrophy, and a number of other clinical parameters. The vast majority of individuals are seen by specialists who keep detailed records, these will be used to select suitable candidates for the trial. It must be stressed that this is only a safety trial and there will be no therapeutic benefit for those participating.

How long will it be before it is known whether ‘molecular patches’ can be used as a treatment for Duchenne muscular dystrophy?

This research project, if funding is obtained, will run for four years. The first two and a half years will be spent in optimising the patches and working on delivery mechanisms. The third year is likely to see the preparation for the clinical trial, which will take place towards the end of year three and into year four. It is unlikely that a definitive answer as to its effectiveness in humans will be known before the end of a four-year period. The Consortium will release information throughout the duration of the project, as results from animal model studies are available.

Are ‘molecular patches’ a cure?

No, this type of therapy is not a cure because the faulty dystrophin gene is still present. This means that if proven to be effective, this treatment would need to be repeated and how often this would need to be done will become apparent during this project.

Will this trial definitely take place?

At present this application is with the DoH and the outcome is likely to be known towards the end of March.

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