making muscle wasting history

This is a summary of the article written by Aurelie Goyenvalle, Jane T Seto, Kay E Davies and Jeffrey Chamberlain, which was published by Oxford University Press on 5 April 2011.

The article is a review of the current status of various therapies and potential treatments for Duchenne Muscular Dystrophy. At present there is no effective therapy to stop the progression of the disease, although several promising experimental strategies are currently under investigation. These include gene therapy aiming at reintroducing dystrophin to muscle tissues. There are several different routes under investigation, these include the reintroduction of the dystrophin gene using viral vectors, exon skipping and pharmacological approaches (using drugs/medicines). All the therapies face challenges due to the need to target different muscles in the body, including heart and breathing muscles, the need for long term effect, the immune response (rejection) and the problem of fibrosis (scar tissue forming in muscles).

Viral Vector-Mediated Gene Delivery
This approach harnesses the ability of viruses to enter a variety of cell types and deposit their genomes. By using an ‘empty’ virus (viral vector) the virus can be used to deliver new material, such as a shortened version of the dystrophin gene. The idea is to provide an alternative copy of the functional dystrophin gene for patients rather than repair their own gene. One advantage of this method is that it isn’t dependent on any particular mutation in a patient’s gene. However, finding a safe way to deliver a replacement dystrophin gene to all muscles in the body is a huge challenge. One of the main challenges is that this type of treatment can trigger an immune reaction.

There are three types of viral vectors currently being studied, these are: adenoviral, adeno-associated viral (AAV) and lentiviral vectors. All three have shown some success in preliminary tests, however, only a reengineered version of AAV vectors called rAAV has progressed to clinical trials.

Lentiviruses have a relatively large carrying capacity and low toxicity, but have not been able to deliver to muscles throughout the body in animal trials. However this class of vector is the best at correcting stem cells and may prove useful for cell therapy of the muscular dystrophies.

Adenoviral vectors have a much larger carrying capacity and some versions can carry the full length dystrophin coding sequence, however, suppression of the immune system is required for long-term use and there have been toxicity issues. Also, these types of vectors have not been able to achieve bodywide delivery of genes to muscles.

rAAVs have attracted wide spread interest because they are able to target muscle cells particularly well when introduced into the bloodstream. However, the rAAV is only able to carry a small amount of gene material. This is leading to the development of ‘mini-dystrophin’.

Mini Dystrophin
The limited carrying capacity of rAAV vectors means that shortened versions of dystrophin must be used. The development of mini- or micro-dystrophin follows the discovery that some very mildly affected Becker muscular dystrophy patients have large genomic deletions in the dystrophin gene and yet some dystrophin is still produced. Work from several labs has shown that two large regions of dystrophin can be shortened with minimal impact on the effectiveness of the gene.

Immune system responses to gene transfer using rAAV
rAAVs can trigger an immune system response. The concern is that dystrophin itself is triggering the reaction in dystrophin-deficient patients. To help combat this problem, several groups are beginning to use a specific ‘promoter’, or gene on/off switch, to produce dystrophin only in muscle and not in immune cells. Another alternative is to deliver a micro-utrophin instead of the micro-dystrophin in DMD patients. Utrophin is a similar protein to dystrophin, and is present in DMD patients and could be over-expressed to compensate for the lack of dystrophin.

If of the immune reactions to AAV, dystrophin and/or utrophin can be controlled, then the rAAV vector could be a viable method of gene therapy. (NOTE: shortened versions of dystrophin and utrophin have been developed and work quite well).

Exon Skipping
Antisense-induced exon skipping aims to remove the mutated or additional exon(s) which are preventing the gene from being read correctly and so producing dystrophin. This approach has been shown to induce shortened forms of dystrophin. Antisense oligonucleotide (AO) chemistries target specific exons depending on the different genetic mutations.

The principles of exon-skipping were first demonstrated in 1996, and again in 1998. Since then there have been numerous studies that have provided pre-clinical evidence that there this could be a potential treatment.
Following encouraging results from animal trials, there are groups in the Netherlands and the UK working towards clinical evaluation of antisense-mediated exon skipping in DMD patients. The Dutch consortium are working with Prosensa on a compound to skip exon 51 (PRO051). This treatment was well tolerated, showed skipping of exon 51 and the development of dystrophin in the vast majority of muscle fibres at levels between 17% and 35%. The UK based MDEX consortium working with AVI Biopharma, also working on a compound to skip exon 51 (AVI-4658 – which is since been named: Eteplirsen). Results from this trial also show that it was well tolerated and that dystrophin expression was up by approximately 42%.
Both studies have been followed by repeat trials where treatment for the boys was continued. Both report positive results and improvement in the 6 minute walk test.

The potential of exon skipping as a therapeutic strategy for DMD has developed from a plausible notion in the mid-nineties, to the point where early clinical trials show it holds realistic prospects of providing genuine therapeutic benefit. However there are still some hurdles to overcome, some scientific, some regulatory.

Challenges for Exon Skipping
One of the hurdles is the poor delivery of AO to all muscle groups and the relatively rapid clearance from the system. This means that regular repeat treatments through the whole of a patient’s life will be necessary. It has also proven difficult to get the treatment to work in the heart. Recent developments using cell penetrating peptide-conjugated PMO (PPMO) have addressed these issues, however, there are toxicity issues with PPMO. The use of peptides could also trigger an immune response.

Despite the very promising results of the initial trials, there are still major regulatory hurdles before gaining approval. This is because there is a slightly different compound required for each variation of the mutated gene. Under current legislation each variation would have to undergo full clinical trials which are both time consuming and expensive. For some patients with very rare variations of DMD, there may not even be enough patients to test effectively. For this reason, there is work being undertaken to see if the FDA would be prepared to view AOs as a class of drugs, with multiple variations. This would be a first for the FDA, but one that needs to be addressed for the future if personalized genetic medicines are to become a reality.

Another challenge for exon skipping is that the effect of the therapy does not last long, so repeat treatments will be necessary. An alternative to weekly or monthly injections is to deliver the AO using viral vectors as discussed at the beginning of this article. There are currently studies looking at this. This type of approach could be combined with cell-based therapies using lentiviral vectors. This could be effective at targeting multiple exons, and could be a good approach to the delivery of personalized medicines.

Pharmacological Approaches

Utrophin Upregulation
A promising pharmacological treatment for DMD aims to increase levels of utrophin, to compensate for the absence of dystrophin. Drug-based utrophin therapy has many advantages as it should be effective for all DMD patients, regardless of the specific gene defect. Also too much utrophin does not seem to cause a reaction in cells other than muscles (unlike the immune reaction that may occur with dystrophin).

BioMarin Pharmaceuticals has identified a small molecule (known as BMN195 and SMT1100) that has recently been tested and while it showed promising utrophin upregulation potential in pre-clinical trials, it could not reach effective levels during the clinical study. There are no safety issues with BMN195/SMT1100, so work is now being undertaken to reformulate the drug for future trials.

Read-through strategies for suppression of nonsense mutations
Some mutations of the dystrophin gene mean that there is a premature ‘stop’ sign, which prevents dystrophin being produced. Research is currently looking for a drug based approach to effectively remove the ‘stop’ sign, so that the full gene is read and dystrophin is produced. Two drugs, gentamicin and ataluren (also known as PCT124) have produced promising results, but the most recent trial has not demonstrated conclusively that it works. This could be due to the design of the trial. This is prime example of just how important the design of clinical trials are.

Conclusion
Since the cloning of the dystrophin gene almost 25 years ago, DMD has gone from a disorder viewed often as incurable or hopeless to one with numerous potential treatment options. From a genetic standpoint, methods to increase dystrophin or utrophin production are showing great promise. Despite this potential there is still much work to be done before we have a successful treatment. This work will comprise of a fine balance between animal studies and human clinical trials, favouring those therapies that show safety and promising results in humans, while other those that are less successful are deprioritised.

As the most successful therapies are found and fine tuned, we expect to see a gradual development of new therapies over the coming years that will increasingly extend lifespan and improve the quality of life for patients with all forms of muscular dystrophy.