Myospreader improves gene editing in skeletal muscle by myonuclear propagation.

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.

Myospreader improves gene editing in skeletal muscle by myonuclear propagation.

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.

Molecular characterization of myotonic dystrophy fibroblast cell lines for use in small molecule screening.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are common forms of adult onset muscular dystrophy. Pathogenesis in both diseases is largely driven by production of toxic-expanded repeat RNAs that sequester MBNL RNA-binding proteins, causing mis-splicing. Given this shared pathogenesis, we hypothesized that diamidines, small molecules that rescue mis-splicing in DM1 models, could also rescue mis-splicing in DM2 models. While several DM1 cell models exist, few are available for DM2 limiting research and therapeutic development. Here, we characterize DM1 and DM2 patient-derived fibroblasts for use in small molecule screens and therapeutic studies. We identify mis-splicing events unique to DM2 fibroblasts and common events shared with DM1 fibroblasts. We show that diamidines can partially rescue molecular phenotypes in both DM1 and DM2 fibroblasts. This study demonstrates the potential of fibroblasts as models for DM1 and DM2, which will help meet an important need for well-characterized DM2 cell models.

Transcriptome alterations in myotonic dystrophy frontal cortex.

Myotonic dystrophy (DM) is caused by expanded CTG/CCTG repeats, causing symptoms in skeletal muscle, heart, and central nervous system (CNS). CNS issues are debilitating and include hypersomnolence, executive dysfunction, white matter atrophy, and neurofibrillary tangles. Here, we generate RNA-seq transcriptomes from DM and unaffected frontal cortex and identify 130 high-confidence splicing changes, most occurring only in cortex, not skeletal muscle or heart. Mis-spliced exons occur in neurotransmitter receptors, ion channels, and synaptic scaffolds, and GRIP1 mis-splicing modulates kinesin association. Optical mapping of expanded CTG repeats reveals extreme mosaicism, with some alleles showing >1,000 CTGs. Mis-splicing severity correlates with CTG repeat length across individuals. Upregulated genes tend to be microglial and endothelial, suggesting neuroinflammation, and downregulated genes tend to be neuronal. Many gene expression changes strongly correlate with mis-splicing, suggesting candidate biomarkers of disease. These findings provide a framework for mechanistic and therapeutic studies of the DM CNS.