Myonuclear domain-associated and central nucleation-dependent spatial restriction of dystrophin protein expression in a novel DMD mouse model

Chwalenia, K.; Feng, V.-Y.; Hemmer, N.; Hildyard, J. C. W.; Roskrow, L. E.; Piercy, R. J.; Wang, E. T.; Aartsma-Rus, A.; van Putten, M.; Wood, M. J. A.; Roberts, T. C.

doi:10.1101/2025.07.24.666562

bioRxiv · 10.1101/2025.07.24.666562

Myonuclear domain-associated and central nucleation-dependent spatial restriction of dystrophin protein expression in a novel DMD mouse model

Chwalenia, K.·Feng, V.-Y.·Hemmer, N.·Hildyard, J. C. W.·Roskrow, L. E.·Piercy, R. J.·Wang, E. T.·Aartsma-Rus, A.·van Putten, M.·Wood, M. J. A.·Roberts, T. C.

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Abstract

The restoration of uniformly-distributed dystrophin protein expression is an important consideration for the development of advanced therapeutics for Duchenne muscular dystrophy (DMD). To explore this concept, we generated a novel genetic mouse model (mdx52-Xist{Delta}hs) that expresses variable, and non-uniformly distributed, dystrophin protein from birth as a consequence of skewed X-chromosome inactivation. mdx52-Xist{Delta}hs myofibers are heterokaryons containing a mixture of myonuclei expressing either wild-type or mutant dystrophin alleles in a mutually exclusive manner, resulting in dystrophin protein being spatially restricted to corresponding dystrophin-expressing myonuclear domains. This phenotype models the situation in female DMD carriers, and dystrophic muscle in which dystrophin has been incompletely restored by partially-effective experimental therapeutics. Total dystrophin expression increased in aged (60-week-old) mdx52-Xist{Delta}hs mice relative to 6-week-old adults, suggestive of an accumulation of dystrophin-expressing myonuclei through positive selection, although this was insufficient to resolve sarcolemmal dystrophin patchiness. Nevertheless, compared to mice expressing no dystrophin, non-uniformly-distributed dystrophin was protective against pathology-related muscle turnover in an expression-level-dependent manner in both adult and aged mdx52-Xist{Delta}hs mice. Systematic classification of isolated mdx52-Xist{Delta}hs myofibers revealed profound differences associated with central nucleation, with dystrophin found to be translationally repressed in centrally-nucleated myofibers and myofiber segments. These findings have important implications for the development of dystrophin restoration therapies.

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Chwalenia, K., Feng, V.-Y., Hemmer, N., Hildyard, J. C. W., Roskrow, L. E., Piercy, R. J., Wang, E. T., Aartsma-Rus, A., van Putten, M., Wood, M. J. A., Roberts, T. C.. 2025-07-24. Myonuclear domain-associated and central nucleation-dependent spatial restriction of dystrophin protein expression in a novel DMD mouse model. https://doi.org/10.1101/2025.07.24.666562

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