In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: Therapeutic Strategies for Dystrophin Replacement in Duchenne Muscular Dystrophy
We start with a review from Frontiers in Medicine on dystrophin replacement by Happi Mbakam et al to get back up to speed. The review gives a nice comprehensive overview of different dystrophin replacing techniques but also contains some mistakes. Doi 10.3389/fmed.2022.859930.
The authors explain that Duchenne is caused by lack of dystrophin and that restoring dystrophin has the final expectations of a cure. I disagree – restoring dystrophin will slow down progression or stop it but not undo accumulated damage (so no cure).
Authors explain about dystrophin structure and function: the actin binding domain binds the cytoskeleton and the cysteine rich domain binds to the extracellular matrix. In between the rod domain gives flexibility and binds lipids and microtubuli.
The C-terminal (i.e. everything after the cysteine rich domain) binds many proteins and protein complexes. Thousands of different mutations have been reported, mostly deletions of 1 or more exons. Authors indicated that the frequency of deletions varies for different regions with higher frequencies in Africa (88%) than elsewhere (64-72%). My take on this is that in Africa due to limitations in diagnostic techniques primarily deletions are picked up (can be done with a multiplex PCR), while duplications and small mutations are missed.
It is possible the frequencies of mutation types vary, but I know that often in Africa MLPA and small mutation analysis are not available. If you cannot look you won’t find these mutations. Authors give an overview of different strategies to restore dystrophin.
Stop codon readthrough. This applies only to nonsense mutations (where a single base pair substitution changes an amino acid code into a stop code). Gentamicin, an antibiotic, is a potent suppressor of stop codon readthrough, but has side effects that preclude long term use.
Authors also discuss PTC Therapeutics’ Ataluren as an alternative and summarize the completed and ongoing clinical trials. They fail to mention however, that Ataluren has been conditionally approved by the EMA for Duchenne patients with nonsense mutations since 2014.
Authors give a very comprehensive summary of all exon skipping trials (22 clinical trials!). As mentioned before, 4 exon skipping compounds are approved by the FDA: eteplirsen, casimersen, viltolarsen and golodirsen (for skipping exons 51, 45, 53 & 53).
Treatment results in low levels of dystrophin. Some of the ongoing trials aim to assess if these levels of dystrophin improve functional decline, while others try to improve the efficacy of exon skipping compounds.
Authors also highlight the use of a more long term exon skipping, using viral vectors (AAV) to deliver a gene that induces exon skipping (U7 or U1 snRNP). The exon 2 skipping trial from Kevin Flanigan is going well. 3 patients treated so far
All showing restoration of full length dystrophin at variable levels, with the youngest patient (<1 year old at time of treatment) showing very high dystrophin levels. Astellas aimed to develop this approach for other exons. However, they announced in April that this work will be stopped. Authors do not mention this, but the paper was published in March so there was no way they could of course. Authors give a very nice overview of different genome editing approaches.
How they work, what the pros and cons are. Methods relying on deletions are more sensitive to off target edits and insertion events when e.g. AAV is used to deliver components. Base and prime editing do not cause a double stranded break but are currently less efficient.
For gene replacement, authors focus on micro-dystrophin AAV: there is micro-dystrophin expression in each of the trials, but it is not yet clear if this reduces disease progression AND there are severe side effects seen in some patients, including the sad death of one patient.
Authors also outline other approaches to try and restore full length dystrophin, e.g. using a human artificial chromosome. This avoids the chance of insertion mutagenesis. However, delivery and instability of these chromosomes for now preclude clinical development.
Another way to deliver dystrophin is by transplanting muscle stem cells from healthy donors. Sadly, myoblasts do not proliferate well in culture dishes and also do not migrate far from the injection site, even after local injections.
Authors finally discuss utrophin upregulation (trials stopped due to lack of efficacy) and givinostat treatment (which targets DNA modifications but does not restore dystrophin so I do not understand why this is part of the review).
Authors conclude that there is still a lot to be done and learn for each of the approaches. I agree. And while there are some minor errors and oversights in the review, I commend the authors for giving a comprehensive overview that explains well and is not overly long.