#apaperaday: Severe cardiac and skeletal manifestations in DMD-edited microminipigs: an advanced surrogate for Duchenne muscular dystrophy
In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: Severe cardiac and skeletal manifestations in DMD-edited microminipigs: an advanced surrogate for Duchenne muscular dystrophy
Today’s pick is from Communication Biology by Otake et al on a Duchenne microminipig model. I was alerted to this by Yoshi Aoki at #AOMC2024 in Nara. DOI: 10.1038/s42003-024-06222-5
Authors introduce there are many animal models for Duchenne, but most of them have anatomic and physiological differences with humans. Pigs are closer than e.g. rats, mice and dogs. However, pigs are big, even minipigs weigh 200 kg, which makes research very expensive.
Another challenge is that Duchenne minipig models have a very severe disease and most die shortly after birth. Authors here propose microminipigs as an alternative. These are only 25 kgs as adults. Authors set out to make a Duchenne microminipig model using gene editing.
They designed a single guide RNA targeting pig dystrophin exon 23, based on the mdx mouse mutation in exon 23. The system worked in cultured cells, so then authors used it in embryos, resulting in 30 potentially edited embryos that were transferred in a foster pig.
3 piglets were born, which had a deletion in exon 23 in some of their cells. 1 of the piglets also had the mutation in its sperm, which was harvested at 8 months and used to fertilize a healthy sow. This resulted in 4 female piglets that carried the exon 23 deletion on 1 allele
Using these carriers, male offspring with the mutation was born, which were further analyzed: on RNA, the deletion was confirmed (a 11 nucleotide deletion, which is out of frame). Western blotting and immunofluorescence analysis revealed that the piglets had no muscle dystrophin
Dp71 could be detected (as expected, given that the deletion is in exon 23, while this isoform starts in intron 62/exon 63). Utrophin was upregulated in skeletal muscle. Functionally, the pigs showed muscle weakness, walking problems and a slower gait from an early age.
From 3 months of age, the pigs also developed respiratory problems and swallowing issues. The ejection fraction became reduced at 6 months, and worsened at 12 months. Serum creatine kinase levels and other muscle damage markers were elevated.
A few piglets suddenly died < 6 months, likely due to dyspnea (breathing problems). On histology there were signs of muscle damage, necrosis and regeneration already at 2 months. Things progressed with time. Heart was OK at 2 months, but fibrosis started at 6 months
The maximum survival was 30 months, when cardiac histology and pathology was worse. The brain and other organs showed no overt signs of pathology. Ejection fractions were measured and starting 6 months it was slightly reduced (60% vs 52%), and worsened with time.
Authors discuss that the symptoms of the microminipigs are similar to those of the Duchenne dog models. They outline that it is not clear what caused the sudden death in 6 of the pigs, but likely it was diaphragm dysfunction. In older pigs cardiomyopathy became apparent.
While no overt signs of brain pathology was detected, this does not mean the pigs have no brain problems. For Duchenne patients there are also no overt anatomical changes, while detailed analysis (not yet done in the pigs) does reveal changes, underlying cognitive issues.
This is a line of research that still needs to be done in the pig (I would be interested to see whether they have difficulties with learning and how their behavior is). Authors compare their microminipig to other pig models, which often die much earlier.
The fact that this model is small and survives longer makes it easier to do research and to test therapies in the future. Authors clearly outline that more work is needed, with longer term studies and additional studies in various aspects of pathology and therapeutic studies.
I appreciate the authors sharing this work and I think the model can be useful – also to study cardiomyopathy due to dystrophinopathy. However, what I do not understand is why the authors made the mutation in exon 23 rather than in the mutation hotspot.
The mdx mouse is a spontaneous model, so we had no choice in the location of the mutation (outside of the mutation hotspot). However, when making a model, you can chose. Having e.g. a deletion of exon 52 would be more useful for mutation specific therapy research
e.g. skipping exon 23 will restore dystrophin in this pig model, but that dystrophin will have a different functionality than Duchenne patients have after skipping exon 51/53/45 etc. Studying a more translationally relevant mutation would be more interesting for me personally.
