- Mutation-specific approaches
| PTC124 and gentamicin | |
| Application: | These drugs only work for patients with a "stop signal" mutation. This is the case for ~15% of Duchenne patients. The drugs can also be beneficial for individuals with stop codons in other genes (e.g. cystic fibrosis patients). |
| Aim: | To force the cell to ignore the mutated stop codon and produce a complete dystrophin protein. |
| Background: | All genes have a start signal and a stop signal so the machinery that translates genes into proteins knows where to begin and where to end. Sometimes a small mutation can introduce a stop signal within the gene (in addition to the one at the end). Normal stop signals generally differ slightly from these mutated stop signals (compare it to a stop signal at a busy intersection (normal stop signal) and one on a highway (mutated stop signal). Nevertheless, the cell will follow the stop signal and will stop the translation of the protein prematurely. There are drugs that suppress the usage of these mutated stop codons, while they do not effect the normal stop codons. The first drug identified to do this in cultured cells and Duchenne mouse models was gentamicin (an antibiotic of the animoglycoside class). |
| Clinical trial: | Gentamicin has been tested in Duchenne patients, but has never convincingly shown dystrophin restoration. |
| Challenge 1: | In addition to its low efficiency, gentamicin is toxic when used for longer periods (it can damage the ears and the kidney). |
| Solution 1: | Screening a large number of drugs resulted in the identification of a drug that was also able to force cells to ignore mutated stop codons, without the toxic side effects. This drug is called PTC124 or ataluren® and is developed by PTC Therapeutics (USA). It can be taken orally and resulted in dystrophin restoration in cultured cells and the mdx Duchenne mouse model. |
| Clinical trials: | PTC124 was safe in healthy volunteers. A first trial in Duchenne patients has been performed. Patients received different doses of PTC124 daily for 4 weeks. Treatment was tolerated well by patients and restored dystrophin clearly at highest doses. Trials to test whether this also results in functional improvement a longer clinical trail have been performed in multiple centers in the USA and Europe. Unfortunately, treatment did not convincingly lead in to a functional improvement when compared to placebo treated patients using a 6 minute walk test and therefore the trials have been put on hold. It is not yet known whether PTC124 treatment resulted in dystrophin restoration (and if so at what levels) in these patients. PTC is currently performing a full analysis on all data of the participating patients and will keep patients updated of these results please visit either the PTC website or the Parent Project Website for further details. |
| Link to preliminary results | |
| Exon skipping | |
| Aim: | To correct the genetic code and allow the production of a partially functional dystrophin. |
| Background: | The genetic code of genes is dispersed over so called exons. When a protein needs to be made, genes make a temporary copy (called RNA). Before this RNA can be translated into protein the exons first need to be joined and the intermittent pieces that do not contain the genetic code (introns) need to be removed. This is a process that is called "splicing". |
| In Duchenne patients the genetic code of the dystrophin gene is disrupted, leading to premature truncation of the translation from gene into protein. In Becker patients mutations maintain the genetic code, allowing for a protein that maintains the functional domains. | |
| Exon skipping aims to restore the genetic code from Duchenne patients, so a partially functional, Becker-like dystrophin protein can be made, rather than a non functional Duchenne protein. This is achieved by AONs (antisense oligonucleotides). AONs are small pieces of modified RNA that recognize a target exon, bind to it and hide it from the splicing machinery. This results in the skipping of said exon and restoration of the genetic code. | |
| AON treatment has induced exon skipping resulting in the production of Becker-like dystrophins in patient-derived cultured cells and the mdx mouse model. In the mouse model, this was accompanied by functional improvement. | |
| There are different types of AONs (chemistries). | |
| Applicability: | For different mutations and types of mutations different exons need to be skipped to restore the genetic code. As most patients have a deletion and these cluster in a hotspot, the skipping of some exons applies to more patients than others. A much more comprehensive discussion of exon skipping, including images that help to visualize the way it works, is available here. |
| While exon skipping would be beneficial to the majority of mutations, there are some exceptions. | |
| Challenge 1: | Exon skipping cannot be tested in healthy volunteers, since skipping an exon that restores the genetic code in patients, disrupts the genetic code in patients (thus potentially converting them in Duchenne patients). |
| Clinical trials: | The first clinical trial tested local injection of exon 51 AONs in the shin muscle 4 Duchenne patients (Prosensa and LUMC). Dystrophin was restored locally in each patient. |
| A second trial using a different chemistry took place in London (Muntoni/AVI biopharma). | |
| Challenge 2: | Over 30% of the human body consists of muscle, so it is impossible to perform local injections of each and every muscle. |
| Solution 2: | As a result of the disease, patients and mouse muscles are leaky (damaged). While AONs are not taken up from blood by healthy muscles, they are by the leaky damaged muscle. Thus in this case, the disease helps the treatment. Intravenous and subcutaneous injections in mouse models have resulted in body wide exon skipping and dystrophin restoration. |
| Clinical trials: | Clinical trials are ongoing where different doses of 2OMePS (Prosensa Therapeutics) or PMO (AVI-Biopharma) AONs are tested through subcutaneous injection or intravenous injection, respectively. Final results are pending, but both 2OMePS and PMO treatment appears to result in dystrophin restoration in the absence of toxic effects! The primary objective of these trials is to confirm that the AONs are safe. Longer trials, involving more patients and a placebo group are needed to confirm whether the dystrophin restoration also leads to improved muscle function. |
| Challenge 3: | AONs to skip different exons are considered different drugs by the regulatory agencies. This means that developing AONs for different exons is very costly and time consuming, as each has to go through all stages of preclinical and clinical development. |
| Solution: | Hopefully, AON development will become faster after the first 2 or 3. TREAT-NMD is coordinating a discussion about this with regulatory agencies on behalf of exon skipping scientists, clinicians and industry. |
| Clinical trials; | Prosensa recently started a phase I/II trial where different doses of 2OMePS AONs targeting exon 44 are tested. |
