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Generation of a Dystrophin Mutant in Dog by Nuclear Transfer Using CRISPR:Cas9-Mediated Somatic Cells- A Preliminary Study

#apaperaday: Generation of a Dystrophin Mutant in Dog by Nuclear Transfer Using CRISPR/Cas9-Mediated Somatic Cells: A Preliminary Study

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: Generation of a Dystrophin Mutant in Dog by Nuclear Transfer Using CRISPR/Cas9-Mediated Somatic Cells: A Preliminary Study

Today’s pick is from International Journal of Molecular Sciences by Oh et al on crispr mediated generation of a dystrophic dog model. Doi 10.3390/ijms23052898

Authors start by explaining why dogs are a good model system for studying human disease, as they are more similar than e.g. mice and since they share a living environment with humans. I am not very convinced about this argument given the ethical issue of using dogs.

Authors describe spontaneous animal models for Duchenne like mice (mdx) & dogs (golden retriever muscular dystrophy). Mice are less severely affected than humans & preclinical studies translate poorly (I would argue partly also because of poor studies not only due to mouse model)

Dogs are more severely affected. However, authors do not describe is that in the GRMD model there is a lot of variability in disease progression that is not predictable at an early stage. This makes preclinical studies challenges as group sizes should be larger than with mice.

In either case, authors want to make the statement that having another dog model for Duchenne would be a good idea and they are making it with genome editing targeting exon 6…I think this is an example of ‘not everything that can be done is a good idea’.

Exon 6 is located in minor deletion hotspot (authors claim it a common location of mutations no so: exon 45-55 is much more common for humans AND a naturally occurring mutation in this region has occurred in a dog model that is currently assessed.

Authors generated a 57 base pair deletion within exon 6 causing a frame-shift. Authors do not study if the internally deleted exon 6 is included in mRNA, only do protein analysis. It is possible the exon is skipped. Regardless of whether it is skipped, reading frame is disrupted

However, if exon 6 is skipped, exon 7 and 8 skipping are needed to restore the reading frame, while if it is included exon 6, 7 and 8 skipping are required. Not the best model for exon skipping approaches, but authors want to use it for genome editing they explain.

Generating the model was challenging. Authors used somatic cell nuclear transfer as this results in a full knockout rather than a chimera with zygote editing. They generated 26 reconstructed oocytes, resulting in only a puppy. The paper is the preliminary analysis of this animal

Authors show that CK is increased, especially after exercise – this is as expected for a dystrophic model. ECG is normal at 5 months, but some minor change in the Q wave was apparent. MRI showed increased fat in the rectusrector femoris and atrophy of some muscles.

Also in Duchenne patients some muscles are affected earlier than others – however, this was also known already from existing Duchenne dog models. After 6 months a biceps biopsy was taken. H&E showed some necrosis and central nucleation. No fibrosis staining was done alas.

Authors also stained for dystrophin and utrophin. I do not think this dog fully lacks dystrophin – there are clear trace amounts visible. On Western blot also traces can be observed. Utrophin expression is increased but dystrophin not fully absent

Authors describe preliminary functional studies in the discussion – I think these should have been part of the results. Apparently there is a gait difference and the dog  became reluctant to exercise at 10 months. Authors are not sure whether the dog will lose ambulation.

Authors do not discuss that dogs walk on 4 legs, and other dog models without dystrophin do not lose ambulation. Authors claim that because the MRI findings of the young dog are similar to those in young Duchenne patients, it is likely the disease will progress similarly.

There is of course no guarantee for this. Authors did not look into other aspects known to affect GRMD, such as swallowing difficulties and tongue problems. They indicate longer term follow up with be needed and that their dog will be useful for genome editing therapy development

I agree that longer term follow up is needed. However, this is one dog and given its severity it is unlikely to breed. Authors would have to generate new dogs – with a very low efficiency – for additional studies. Genome editing studies will likely focus on major hotspot at that

Plus genome editing studies can be done to achieve proof of concept in a larger model than mouse or rat. However, the development requires a humanized model as due to species differences the guides are likely species specific (as are off target effects).

Now that genome editing is relatively straightforward I think there is a risk of animal models being generated just because it is possible, not because they are needed. In the past, it was so laborious you only did it if you had a very good cause and it was worth the effort.

Now it is easier and one may think ‘let’s just try’. I do not think that aligns with the 3Rs and hope people will resort to cell model systems when possible (because genome editing has a lot to offer) rather than just generate another animal model and see where it gets them.


Pictures by Annemieke, used with permission.