Molecular Patch Therapy Q & A - 07/05

'Molecular Patches' as a therapy for Duchenne muscular dystrophy: A clinical research trial funded by the Department of Health.

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

You can download this Q & A as a PDF: Molecular Patches - Q&A - 07/05.pdf (36 kb) [pdf]

Contents:


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

A: 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.

Q: What is the difference between Duchenne muscular dystrophyand Becker muscular dystrophy?

A: In Duchenne muscular dystrophy, there is a total or nearly total absence of functional dystrophin protein. In Becker muscular dystrophy, a shortened dystrophin protein is produced, which is partially functional, which means the disease is less severe. The severity of weakness in Becker muscular dystrophy can vary, but most Becker muscular dystrophy patients are able to walk in adulthood.

Q: How are proteins made?

A: 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:

 A sentence that reads THEWTOMMANATIANDPIQHISURDIDOGITRANOVINFORTRIATHEDOOPIBUS, used to show how patches can be used to skip faults in the gene

 

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 as shadow areas), 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



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

A: 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:
A sentence that reads THE MAN AHI SDO GRA NFO RTH EBU S as the 'ND' has been removed after the second A

 

 

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.



Q: What happens in Becker muscular dystrophy?

A: 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:A sentence that reads THE MAN RAN FOR THE BUS after the words AND HIS DOG have been removed

 

 

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.



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

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

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

A: 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.



Q: Does this really work?

A: 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.

Q: Do scientists understand how this works in the body?

A: 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.



Q: Will it work for everyone with Duchenne muscular dystrophy?

A: No, but it is thought that around 60-70% of the genetic errors associated with Duchenne muscular dystrophy could be treated with the use of ‘molecular patches’.



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

A: This technique is just one of many identified by scientists as having therapeutic potential. Researchers worldwide 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.

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

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

Q: Will the same ‘molecular patch’ work for everyone?

A: 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’.

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

A: 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.

Q: I have heard about a therapeutic trial funded by the Department of Health on “molecular patches”. What does this involve?

A: The three charities involved in Duchenne muscular dystrophy in the UK are the Muscular Dystrophy Campaign, Parents Project UK (PPUK) and Duchenne Family Support Group. Together with a group of scientists they succeeded in securing funds towards a gene therapy trial in Duchenne muscular dystrophy using “molecular patches”.

The aims of this project are to:

  • Optimise the structure of these “molecular patches”
  • Improve delivery techniques so that these patches could eventually be administered to many muscles at once
  • Test the efficacy of these ‘molecular patches’ in various models of the disease
  • Perform a study in a small group of Duchenne muscular dystrophy children to test the safety of the ‘molecular patch’ administered to a muscle after an injection and see whether functional dystrophin is produced.

    Q: Who will participate in the initial safety trial?

    A: Participants will be selected using specific inclusion criteria. These will include age and the type of genetic error causing Duchenne muscular dystrophy. A number of other clinical parameters will be taken into account such as the ability of the patients and the family to consent and collaborate with the assessment protocol. Patients with severe cardiomyopathy or respiratory insufficiency (patients ventilated at night) will be excluded for the study.

    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.

    Q: How long will it be before it is known whether ‘molecular patches’ can be used as a treatment for DMD?

    A: This research project is funded for four years. The first two years will be spent in optimising the patches in the laboratory by testing their compatibility and effectiveness with human Duchenne muscular dystrophy cells such as muscle or skin cells and working on delivery mechanisms. At the same time, work will be done towards selecting the best-preserved foot muscle, which will be later on targeted with the ‘molecular patches’. 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. The Consortium will release information throughout the duration of the project

    Q: Are ‘molecular patches’ a cure?

    A: 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.

    Q: How might children with Duchenne muscular dystrophy and their families be asked to help in the future with these studies?

    A: There are a number of different ways in which families may be asked to help in the preparatory work for the clinical trial.

    • Some families may be asked to give permission for a small muscle and/or skin biopsy from their child to be obtained at the time of the diagnosis. This could be studied to help develop ‘molecular patches’ in the laboratory.
    • Some older children have surgery (under general anaesthesia) on their feet or back. Some of these children may be asked to give permission for us to study the appearance of their muscles using different techniques. This will involve the following tests:

    Before the operation: an MRI scan (a special scan) of the children’s feet will allow us to check which muscle would be the best to use to administer the ‘molecular patches’ in the clinical trials. 

    During the operation and whilst asleep in theatre: A very small piece of muscle (the size of an orange pip) will be taken from the top of the child’s foot. This will be studied under a microscope to see what the muscle cells look like. We will also try to use some of the muscle cells to see how they react to the ‘molecular patches’ in the laboratory. A small piece of skin (size of a matchstick) will also be taken from the site of the surgery, so that the skin cells can be grown in cultures and studied to understand how they react to the molecular patches in the laboratory. A blood sample (about two teaspoons) may also be taken.



    Glossary:

    Animal models: Animals having conditions comparable to humans, which can be used to study disease processes. Animals represent a simpler system than humans for studying the roles of different proteins and for testing potential therapies. Results can then be applied to humans, the use of a simpler system often allows quicker progression of research.

    Becker muscular dystrophy: A milder variant of Duchenne muscular dystrophy. It is X-linked, slowly progressive, causes muscle weakness and usually only affects boys.

    Deletion: The loss of a bit of genetic material from a chromosome or gene.

    DNA: Deoxyribonucleic acid, the chemical composition of genes. It contains coded information, arranged in a linear sequence. Each cell’s chromosomes contain about two metres of DNA, yet it is so thin that it is barely visible even with the most powerful microscope. If all the DNA in a human body were stretched end to end it would be long enough to reach the moon and back about 10,000 times.

    Duchenne muscular dystrophy: A genetic disorder, which causes progressive muscle weakness as the muscle cells break down and are eventually lost. Usually it affects only boys and is caused by a lack of dystrophin protein.

    Dystrophin: The protein, which is missing in boys with Duchenne muscular dystrophy and reduced in boys with Becker muscular dystrophy. Dystrophin binds to other proteins in the dystrophin-glycoprotein complex (DGC), absence of these components are implicated in different forms of muscular dystrophy.

    Genes: The coded instructions that govern the make-up of every human being. Genes are made of DNA. Each gene carries instructions for the production of a specific protein. Genes usually come in pairs, one copy inherited from each parent. They are passed on from one generation to the next, and are the basic units of inheritance. Alterations in genes (mutations) can cause inherited disorders.

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