Myospreader improves gene editing in skeletal muscle by myonuclear propagation

Poukalov, K.; Valero, M. C.; Muscato, D.; Adams, L.; Chun, H.; Lee, Y. i. T.; Andrade, N.; Zeier, Z.; Sweeney, H. L.; Wang, E. T.

doi:10.1101/2023.11.06.565807

bioRxiv · 10.1101/2023.11.06.565807

Myospreader improves gene editing in skeletal muscle by myonuclear propagation

Poukalov, K.·Valero, M. C.·Muscato, D.·Adams, L.·Chun, H.·Lee, Y. i. T.·Andrade, N.·Zeier, Z.·Sweeney, H. L.·Wang, E. T.

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Abstract

Successful CRISPR/Cas9-based gene editing in skeletal muscle is dependent on efficient propagation of Cas9 to all myonuclei in the myofiber. However, nuclear-targeted gene therapy cargos are strongly restricted to their myonuclear domain of origin. By screening nuclear localization signals and nuclear export signals, we identify "Myospreader", a combination of short peptide sequences that promotes myonuclear propagation. Appending Myospreader to Cas9 enhances protein stability and myonuclear propagation in myoblasts and myofibers. AAV-delivered Myospreader dCas9 better inhibits transcription of toxic RNA in a myotonic dystrophy mouse model. Furthermore, Myospreader Cas9 achieves higher rates of gene editing in CRISPR reporter and Duchenne muscular dystrophy mouse models. Myospreader reveals design principles relevant to all nuclear-targeted gene therapies and highlights the importance of the spatial dimension in therapeutic development.

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Poukalov, K., Valero, M. C., Muscato, D., Adams, L., Chun, H., Lee, Y. i. T., Andrade, N., Zeier, Z., Sweeney, H. L., Wang, E. T.. 2023-11-06. Myospreader improves gene editing in skeletal muscle by myonuclear propagation. https://doi.org/10.1101/2023.11.06.565807

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