Written by Dr Sarah Yates, Director of Scientific Affairs

Scientists have already made an artificial gene designed to do the job of the one that is faulty in people with Duchenne md. Their task now is to deliver copies of these ‘dystrophin’ genes efficiently into muscle and to make sure that they stay put, get to work and stay working. At the moment, the most promising way of achieving this goal is by using viruses…

What is a virus?

Viruses come in many forms and cause many different symptoms. They are responsible for colds and flus, warts, polio, herpes, rabies and smallpox to name but a few. A virus consists of some genetic coding (RNA or DNA) surrounded by a protein coat and sometimes a fatty outer coat. They are designed to trick cells into accepting them. The cell supports the virus – thinking that its genetic coding is part of its own coding – much in the same way as a cuckoo tricks a mother bird into feeding it, thinking that the intruder is part of her family. For our purposes, viruses have two very important properties:

  • they are highly efficient at getting into calls;
  • they cause their own proteins to be manufactured.

Aren’t viruses dangerous?

In their unaltered state, viruses are infectious and can be harmful. However, the trick is to alter the virus so we keep the properties that we want ( for example its ability to enter the cell and to get the cell to do its bidding) and remove the characteristics that make the virus dangerous. This means removing those bits of coding that cause symptoms, e.g. cold symptoms, and also the coding that would allow the virus to multiply inside the cell.

Why don’t we want the virus to multiply inside the cell?

A virus can multiply itself millions of times and the new viruses can go on to infect other cells within the body in an uncontrolled way. These viruses have been genetically engineered so the last thing you want to do is infect other people with an artificial virus with unpredictable behaviour.

So, we remove the dangerous element of the virus. What do we add to it?

We use the genetic space that we’ve created to insert artificial genetic coding which will make the virus produce the protein that we want it to produce. In the case of Duchenne md, for example, that protein would be dystrophin; in the case of congenital md it would be merosin; or adhalin for one form of limb girdle dystrophy.

How do the viruses reach all tbe muscle cells in the way we want them to?

The virus is grown in artificial conditions in the laboratory. It is accompanied by ‘producer’ cells and ‘helper’ viruses that enable it to grow and multiply, even though the virus that will carry the gene is incapable of replicating itself in live organisms. This means you can get a lot of identical viruses, each one of which will infect one cell. If we can get enough copies of the virus, in theory, we would have sufficiently high numbers to reach a high proportion of muscle cells, even though the infection can’t spread from one cell to another.

This all seems a very complex way of getting the dystrophin protein into muscle. Couldn’t you just inject the protein directly into muscle?

You don’t just need to get this protein in, you have to insert it in exactly the right place within the cell structure. Cells have their own processes to deal with proteins they manufacture for themselves. If dystrophin was simply injected into muscle it wouldn’t lock into the cell’s own protein processing system. Proteins that aren’t properly processed are left open to being damaged by the chemical environment and will be destroyed.

We hear about ‘off the shelf’ viruses, what are they?

Scientists have thought for a long time that viruses are a good way of moving genetic material into cells. By the time our researchers were ready to try and get artificial dystrophin genes into muscle, viruses had already been developed, but they weren’t specifically designed for muscular dystrophy. Using these ‘off-the shelf’ viruses, high dystrophin levels were achieved, initially in cell culture and animal models. However, there are serious problems with the current viruses:

  1. The viruses trigger the immune system in animals with a mature functioning immune system and they are destroyed, i.e. the viruses have a short life. If you tried to introduce the virus a second time, the immune system would be already alerted and would wipe out the invasion before it had even got going
  2. Most viruses currently available don’t have sufficient capacity to carry the full-length dystrophin gene so a mini-gene has had to be used. This does work but in the long run a full-length gene may prove to be more effective and easier to control.
  3. Viruses being used at present aren’t specialised at infecting muscle. In fact the adinovirus which is often used naturally prefers lung tissue which is why, in an unaltered state, some adinoviruses cause cold symptoms.

What we need a virus which is specifically adapted to target muscle, will avoid the immune system and can carry a full length gene…

Yes. But it’s not just a question of designing an efficient virus; what goes in it needs refining too. For example, different sizes of dystropin gene are being tested, and also different controlling regions that will regulate how much dystrophin is made, where it is made and under what circumstances.

So these viruses are being designed to carry the dystrophin gene. What about all the other genes for other conditions?

If a particular muscle disorder is caused by a genetic fault directly affecting muscle due to deficiency of a protein then, on the face of it, a system which works for Duchenne md could be adapted. The bit that would change would be a man-made gene in the middle. The bit that would stay constant would be the design of the virus itself. The controlling regions may also remain the same.

I’ve heard of adenoviruses and retroviruses. What’s the difference?

These are two of the main types of virus being developed for gene therapy. They each have advantages and disadvantages. Scientists are manipulating both to try and maximise their good points and minimise their drawbacks. For example, the retrovirus is particularly good at embedding itself into the genetic code of the cell so that when the cell naturally divides, the virus does too. This could mean that one treatment would be enough. Its disadvantage is that it can only get into cells which are in the act of division, and this can make efficiency low. Adenoviruses used in gene therapy are more efficient at infecting cells than retroviruses but they don’t persist once the cell has divided.

There is more space to carry artificial genes than in a retrovirus. The dystrophin gene is a particularly big gene so this is important.

Where is virus research for muscular dystrophy being carried out?

The main Muscular Dystrophy Campaign funded laboratories are at Guy’s, Royal Holloway and Charing Cross hospitals under Professors Frank Walsh, George Dickson and Dominic Wells respectively. Researchers at other labs, such as Professor Kay Davies at Oxford, collaborate with these teams.

Has gene therapy using viruses ever worked for muscular dystrophy?

Scientists have managed to produce dystrophin in thc muscle cells of young mice that don’t have dystrophin i.e. they have the mouse equivalent of Duchenne md, and the symptoms of muscular dystrophy have been alleviated. The problem is that the young mouse’s immune system is not sufficiently developed to fight the virus in the same way that a human’s immune system is. So the challenge is to devise ways of by-passing the immune system. Also mice muscles are very small so it is relatively easy to treat one muscle in a mouse, but we will need higher efficiency and a better entry system to treat a human .

How will we get the viruses into muscle?

At the moment scientists are giving thc viruses a head start by injecting them straight into muscle and allowing them to reach individual cells from there. Eventually we want a virus that targets muscle so that we could inject into the blood stream. The viruses would then be carried around the body by the blood, from where they would seek out muscle.

Is there still a long way to go?

There are lots of problems that still need solutions and questions that still need to be answered. But the nature of those problems is very clear and there are already theoretical answers to them. A huge amount of investment is going into gene therapy around the world and not just into md. Internationally renowned scientists are dedicating their careers to this field and highly reputable funding bodies are investing big sums of money in it. This wouldn’t be happening unless they were convinced of long-term results.