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expert reaction to the use of genome editing techniques to treat Duchenne muscular dystrophy in a mouse model

A group of researchers have published in study in the journal Science in which they report the use of CRISPR-cas9 genome editing techniques to treat a mouse model of Duchenne muscular dystrophy.


Prof. Robin Lovell-Badge, Professor of genetics at the Crick Institute, said:

“The work in these three papers, which all support each other, is very exciting and offers prospects for application of the methods to treat DMD patients. The results suggest that this genome editing approach may be much better than other methods of somatic gene therapy that are being considered for DMD. However, there are (of course) a number of issues that would need to be resolved before this could happen. The possibility of off-target events occurring in vivo was not adequately addressed. Such events may be of little or no consequence, but again without a detailed analysis this may worry regulators, and it is not clear that suitable methods to do this are yet available. While introduction of the viral vectors systemically will be necessary for any treatment of patients, the majority of whom will be boys (the dystrophin gene is X-linked), this could inadvertently lead to editing within stem cells of the testis that give rise to sperm. If the patients were to breed, then this may lead to passing on of the edited gene to offspring, and constitute germline gene modification. Even if this is not a problem, the survival of DMD patients to breeding age and beyond, while fantastic, would mean that they might want children who don’t carry the mutation. This might increase the demand for germline gene therapy.

Interestingly, Long et al show in their paper that the same strategy of exon deletion used in their somatic therapy approach could be applied to the fertilised eggs of MDX mice and at an efficiency of 80%, which is remarkably high. Moreover, it is possible in this case to reliably look for off-target events, because the whole mouse is derived from the single cell in which the components were injected as opposed to the millions of cells that have to be infected in somatic gene therapy, and none were found. On the other hand, if one were going to go to the trouble of carrying out germline gene therapy, then it would be preferable to correct the mutation to fully restore dystrophin function. This would require the use of different methods that rely on “homology directed repair” and the inclusion of a DNA template along with Cas9 and gRNA, and currently this is much less efficient than the “non-homology end-joining” mechanism exploited in the somatic gene therapy approach. (N.B. the latter had to be used, because homology directed repair mechanisms do not operate in differentiated muscle cells.)

All three papers use similar somatic genome editing approaches in attempts to restore some dystrophin function in the mdx mouse model of Duchenne Muscular Dystrophy (DMD). They use Cas9 and two guide RNAs (gRNAs) designed to remove the mutated exon 23 from the dystrophin gene. This leads to a slightly smaller dystrophin protein with reduced activity than that present in normal mice, but any activity is considerably better than none. This approach also has the advantage in that it does not require precise correction of the disease-causing mutation: any deletion that prevents splicing of mutant exons and therefore truncation and instability of the protein, or the incorporation of a defective part of the protein, will be sufficient to restore expression of a functional protein. The method could be used, in theory, for many cases of DMD in humans and potentially for other genetic diseases where “exon-skipping” could have a beneficial effect.

Adeno-associated viral (AAV) vectors were used to introduce the Cas9 nuclease and the two gRNAs into postnatal animals, either directly into skeletal muscle, or via the bloodstream such that the viruses might have more systemic effects in cardiac as well as skeletal muscle, and include the diaphragm as well as other critical muscles. All three papers report a low, but significant restoration of dystrophin expressing muscle cells, and of muscle cell function. It is difficult to know whether the degree of restoration seen in the mice, which varies between the studies and from one muscle type to another (generally from about 2-9%) would be sufficient in humans, however, it is thought that as little as 4% of normal dystrophin expression level is sufficient to improve muscle function and 30% protein expression may be sufficient for complete rescue. It may be possible to improve the viral vectors, to make them more efficient and to design them such that they only target the relevant cell type.

Long and colleagues found that the proportion of dystrophin-positive myofibers increases with time, which could reflecting persistent expression of the genome editing components, as they suggest, but it could also be due to improved survival of cells with functional dystrophin compared to un-edited cells. In the case of skeletal muscle, it could also reflect editing within “satellite” stem cells that then could gradually repopulate the muscle with “corrected” muscle cells. This exciting possibility is suggested by data within the Tabebordbar et al paper; although they did not prove this in vivo. However, as there seems to be no equivalent stem cell population in the heart able to give rise to cardiac muscle cells, other methods might be necessary to improve efficiency of the editing in this tissue.”


Prof. Peter Braude, emeritus professor of Obstetrics and Gynaecology, King’s College London, said:

“This is an exciting paper demonstrating in laboratory mice the feasibility of a non-controversial use of the new CRISPR-Cas9 gene editing technology to treat a serious and slowly lethal genetic disease.

The authors used a number of clever modifications of the gene editing technology, such as the use of a more easily packaged smaller molecular scissors nuclease enzyme; the selection of set of guide RNAs to remove but not replace exon 23 which causes premature termination of the dystrophin protein but which still allowed natural function to be restored in a high proportion of animals; and the intravenous administration of the editing tool thus not only addressing the skeletal muscle deterioration which leads to profound disability, but also achieved heart and diaphragmatic muscle improvement which could prevent or ameliorate the respiratory  and cardiac effects which often lead to premature death in DMD patients.

The paper reinforces the powerful nature of gene editing technology, which could be put to good use for the individual affected by the genetic disease, without having to invoke the need for embryonic modification. The fact that that it theoretically has the potential to be used to treat such a large proportion of DMD patients (83%) makes it very exciting indeed.”


Professor Adrian Trasher, Institute of Child Health, GOSH, said:

“This is an exciting and important study demonstrating proof of principle of gene editing in vivo for neuromuscular disease, but still some way to go before translatable to human subjects. A.


Professor Dominic Wells, Royal Veterinary College, said:

“The paper by Nelson and colleagues is an important demonstration that genetic correction can be performed in the mdx mouse model of Duchenne muscular dystrophy (DMD) using a gene therapy delivery system. As noted by the authors, the wide variety of mutations present in DMD means that this approach, similar to antisense mediated exon-skipping, is a patient specific therapy. Some mutations are relatively common such that deletion of exon 51 would treat approximately 13% of all DMD patients. There are also a number of barriers to be overcome before translation to clinical treatment in man. Body wide efficient delivery of an AAV vector has not yet been demonstrated in man. There is also potential for “off-target” side-effects with the CRISPR/Cas9 system and any such effects would not disappear if treatment was discontinued. Thus the current study shows the potential of gene editing in DMD but many further studies will be required before clinical trials could be considered.”


‘In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy’ by C. E. Nelson et al. published in Science on Thursday 31th December. 


Declared interests

Prof. Peter Braude was formerly director of the Centre for Preimplantation Genetic Diagnosis at Guy’s Hospital London, and a member of the HFEA expert panel on Mitochondrial Donation for mtDNA disease.

Prof. Dominic Wells: Member of the British Society for Gene and Cell Therapy, Member of the American Society for Gene and Cell Therapy. Also a member of the Scientific Advisory Board for Akashi Therapeutics, a company developing treatments for Duchenne muscular dystrophy. Currently on Grant panels for Telethon (Italy), AFM (France), MDUK. Previously a grant panel member for BBSRC and NC3Rs. Research grant funding Current and Previous from the BBSRC, MRC, Wellcome Trust, AFM, MDUK and other charities.

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