RESUMO
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.
Assuntos
Sistemas CRISPR-Cas , Núcleo Celular , Edição de Genes , Terapia Genética , Músculo Esquelético , Distrofia Muscular de Duchenne , Edição de Genes/métodos , Animais , Camundongos , Músculo Esquelético/metabolismo , Núcleo Celular/metabolismo , Terapia Genética/métodos , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/genética , Humanos , Sinais de Localização Nuclear/genética , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/genética , Modelos Animais de Doenças , Mioblastos/metabolismoRESUMO
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.
