Researchers, publishing in Science, reported that they were able to restore the expression of dystrophin using genome editing in a canine model of Duchenne Muscular Dystrophy.
Dr Alena Pance, Senior Staff Scientist, Wellcome Trust Sanger Institute, said:
“The paper presents a preliminary study of the use of gene therapy based on Cas9 editing to correct DMD in large mammals. The ability to introduce the gene editing machinery specifically into the targeted cells in a whole organism by systemic injection is very promising towards a potential application of the technology. There are limitations to this study that need to be kept in mind when assessing this potential. Mainly that the number of experimental animals is low so generalisation of the conclusions needs to be careful. Importantly, this research assessed the specificity and degree of the desired editing to confirm genomic changes and dystrophin expression, however this was measured on bulk cell or tissue preparations so the levels of recovery in individual cells is difficult to determine. Finally, the function of the dystrophin expressed was not thoroughly examined and further studies should be performed to verify whether the interaction with other proteins and membranes is correct and the normal function is restored.”
Prof Robin Lovell-Badge, Group Leader, The Francis Crick Institute, said:
“This is a small-scale study, and therefore it must be taken as preliminary, however, it has been carried out very well, and shows great promise as a way to treat patients with Duchenne muscular dystrophy (DMD). It is using small mutations made by genome editing to effectively restore production of the protein that is otherwise missing in many of the muscles of a dog model of DMD that closely resembles the human situation. There are many patients waiting for such methods and data like this will be necessary to persuade regulators to permit clinical trials. These will need to be done with young patients (n.b. they are usually boys because it is an X-linked disease), before their muscles degenerate too much to be restored. This need creates nervousness and further work will be required to improve methods of delivery and to ensure no unwanted side effects, but the results presented in this paper should be seen as very encouraging.
“A substantial proportion of cases of DMD are due to the presence of a mutation in exon 51 that prevents any protein being made. Because the Dystrophin protein contains many repeated blocks (or domains) and only one of these is encoded by exon 51, methods that can lead to this exon being “skipped”, can permit a slightly shorter than normal, but otherwise almost fully functional protein to be made instead. The best previous method to do this, which made use of a small piece of DNA (an oligonucleotide) to confuse the cellular machinery that splices the exons together to make a functional messenger RNA (mRNA), and which is licenced for use in DMD patients, restored dystrophin protein levels to at best 0.32%. By using genome editing methods based on CRISPR/Cas9 to promote exon skipping, the Olson lab had shown previously that this could work to restore active dystrophin protein in both mice and in muscle cells obtained from patient-specific human induced pluripotent stem (iPS) cells. The levels of dystophin made were sufficient to restore muscle function in the mice. However, mice are very small and it was thought that it would be a huge challenge to develop ways to deliver the genome editing components to human patients both safely and with sufficient efficiency to give at least 15% normal levels in all the many muscle cells required to restore normal function. Amoasii and colleagues in the Olson lab decided to test methods in a dog model of DMD, choosing this as an appropriate large animal that would more closely mimic the human situation. They used a virus (AAV9) to deliver the genome editing components to beagle dogs carrying a mutation in exon 50, which is equivalent to mutations occurring in the human exon 51. The virus has an affinity (tropism) to muscle cells, meaning that it will preferentially target these and not other cell types, as well as muscle-specific regulatory regions to drive expression of the Cas9 and a single guide RNA (sgRNA). These will boost efficiencies and hopefully reduce any unwanted side effects.
“The virus was first delivered directly into specific leg muscles of two dogs with DMD. This resulted in about 60% of normal levels of Dystrophin, more than enough to give normal muscle function, within a few weeks. Two other dogs were given the virus in their blood stream. This led to high levels of Dystrophin being made in the heart and in many other muscles throughout the body, although some still showed low levels.
“The authors address some issues relevant to safety, notably they found no off-target mutations. However, they point out that there is still much to be done to prove that the methods will be safe to use. DMD is a horrible debilitating disease, and it might be thought that any treatment that will promote recovery will be better than none. However, if the genome editing approaches are successful when applied to patients, they could well give a permanent “cure”, and restore a normal lifespan rather than the very truncated and difficult one experienced by most sufferers. It will be important that the methods don’t lead to side effects, such as cancer, that would affect them in different, but perhaps equally distressing way. This is why use of large animal models, such as the dogs used in this study, are so important.”
Darren Griffin, Professor of Genetics, University of Kent, said:
“This work represents a small, but very significant step towards the use of gene editing for Duchenne Muscular Dystrophy (DMD). DMD is such a horrible disease, both for the children with it, and also for the families who have to care for them and ultimately watch them die. Any steps towards significant treatment regimes can only be good news. In this study, the use of a large animal model (dog) and the restoration of dystrophin levels to up to 90% are very promising. In the fullness of time, this paper may well be seen as one of the ground breaking studies that led the way to effective treatment.”
Dr Kate Adcock, Director of Research and Innovation at Muscular Dystrophy UK said:
“It is exciting to see advances in how the technique can be applied to Duchenne muscular dystrophy, but as the authors recognise, there are limits to this study. The sample size was small and the study duration too short to know whether the gene editing was safe and effective. Although it seems to have largely boosted dystrophin production, which is key to tackling this condition, the team weren’t looking to record improvements in function. The next step will be to conduct larger, longer-term studies to see if the gene editing approach does help to slow the progression of the condition and improve muscle strength. This won’t be a cure, but that shouldn’t obscure that this is a key step forward in proving the CRISPR/Cas9 technology could work for Duchenne.”
* ‘Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy’ by Amoasii et al. was published in Science on Thursday 30th August.