#apaperaday: Profound cellular defects attribute to muscular pathogenesis in the rhesus monkey model of Duchenne muscular dystrophy

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: Profound cellular defects attribute to muscular pathogenesis in the rhesus monkey model of Duchenne muscular dystrophy

Today’s pick is from @CellCellPress by Ren et al on single cell sequencing of a rhesus monkey model for Duchenne muscular dystrophy DOI: 10.1016/j.cell.2024.08.041

Authors introduce that most of what we know about Duchenne pathology is from mouse models as it is not possible to take longitudinal muscle samples from patients. However, the mdx mouse does not recapitulate pathology, as it is milder than in humans.

You can exacerbate pathology by mutating other genes, but that poses challenges. E.g. if you delete utrophin, the mdx/utrn double knock out is very severely affected. When you test therapies that target pathology you do not know what you treat: lack of dystrophin utrophin or both.

Authors argue non human primates are close to humans and they made a rhesus monkey Duchenne model: with gene editing they made a deletion in exon 5 that resulted in a mosaic monkey with no symptoms (not unexpected as this is similar to female carriers who often have no symptoms).

They used this monkey to generate offspring lacking dystrophin & characterize the model here. Before going into this, a comment: I dont understand why authors made a deletion in exon 5, outside of the hotspot, making it less relevant when testing mutation specific therapies.

This is a similar comment to the one I made yesterday about the microminipig. Making these larger, more complex model is so difficult, that researchers should put in some effort to chose which mutation to generate – also to avoid having to make more models.

Back to the paper: the monkeys generated produced only very low amounts of dystrophin (note out of frame mutations before exon 8 in humans generally lead to a milder pathology and low dystrophin rather than no dystrophin – so again location mutation is suboptimal).

Authors looked into muscle histology of 1 year old monkeys, showing variation in fiber size, centrally located nuclei and fibrosis, which progressed with age. Creatine kinase, ALT and AST (muscle damage markers) were elevated in blood.

Authors also performed functional studies in 1 year old monkeys (about 3-4 year old human equivalent). These monkeys showed a different gate, walked less, hang less and were less strong. Also they used a Gower’s maneuver type of approach when rising from the floor.

The heart was not affected in 1 year old monkeys but respiratory function appeared to already be affected. Authors then performed single cell sequencing on muscle cells (only single nucleated cells, not muscle fibers). Wild type and Duchenne monkeys had the same types of cells.

However, the numbers differed: the Duchenne monkeys had a lot more immune cells and less FAPs (fibro-adipogenic stem cells). The immune cells in wild type monkeys were mainly resident macrophages, while Duchenne monkeys had a series of different immune cells.

Both pro- and anti-inflammatory macrophages and T-cells were detected. Also for the FAPs the subtypes differed: the FAPs of the Duchenne monkeys expressed more extracellular matrix proteins and appeared to differentiate into fibrotic and adipogenic cells.

The muscle stem cells in wild type muscle were mainly quiescent, while in Duchenne monkeys they were activated and proliferating, but appeared to fail to differentiate and some were transdifferentiating also to fibrotic or adipogenic cells.

Authors isolated muscle stem cells from wild type and Duchenne monkeys and confirmed the Duchenne stem cells differentiated less well. TGFbeta was not expressed in muscle stem cells, but was in the inflammatory cells of the Duchenne monkey FAPs.

Authors discuss that it took them years to generate the Duchenne monkeys from the mosaic monkey that was generated first. Authors also discuss the limitations of their study: they only analyzed mononucleated cells, not the muscle fiber. This is something that needs to be done yet.

They also outline that longer term studies are needed – I agree and appreciate the authors outline this (and am of course interested in the results of the future studies). I also appreciate that authors used wild type controls.

I do want to add though that the results authors report here fit well with the pathology model we have for Duchenne, based on a lot of mouse studies and limited analysis of patient samples: inflammation, transdifferentiation of FAPs and failed differentiation of muscle stem cells.