Non-uniform dystrophin re-expression after CRISPR-mediated exon excision in the dystrophin/utrophin double-knockout mouse model of DMD

Hanson, Britt; Stenler, Sofia; Ahlskog, Nina; Chwalenia, Katarzyna; Svrzikapa, Nenad; Coenen-Stass, Anna M.L.; Weinberg, Marc S.; Wood, Matthew J.; Roberts, Thomas C.

doi:10.1016/j.omtn.2022.10.010

Cited by 6 publications

(1 citation statement)

References 86 publications

(143 reference statements)

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“…In contrast, an increased amount of Dp71 variant with an altered C-terminus (Dp71f variant, without exon 78) was observed at the sarcolemma, shown previously to modify the dystrophin function [ 174 ]. Furthermore, nonuniform correction of the DMD gene after the CRISPR/Cas9-mediated exon excision across nuclei in myofibers resulted in poor therapeutic efficacy, despite the overall high dystrophin protein levels, likely because of segmental reparation of the sarcolemma [ 175 , 176 ]. Morin et al indicated that modest but uniform distribution of dystrophin at the membrane of myofibers, obtained e.g., with AONs, might be more therapeutic than high levels of dystrophin generated only in some nuclei in the myofiber [ 175 ].…”

Section: Gene Editing With Crispr/cas9mentioning

confidence: 99%

Dystrophin- and Utrophin-Based Therapeutic Approaches for Treatment of Duchenne Muscular Dystrophy: A Comparative Review

Szwec,

Kapłucha,

Chamberlain

et al. 2023

BioDrugs

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Duchenne muscular dystrophy is a devastating disease that leads to progressive muscle loss and premature death. While medical management focuses mostly on symptomatic treatment, decades of research have resulted in first therapeutics able to restore the affected reading frame of dystrophin transcripts or induce synthesis of a truncated dystrophin protein from a vector, with other strategies based on gene therapy and cell signaling in preclinical or clinical development. Nevertheless, recent reports show that potentially therapeutic dystrophins can be immunogenic in patients. This raises the question of whether a dystrophin paralog, utrophin, could be a more suitable therapeutic protein. Here, we compare dystrophin and utrophin amino acid sequences and structures, combining published data with our extended in silico analyses. We then discuss these results in the context of therapeutic approaches for Duchenne muscular dystrophy. Specifically, we focus on strategies based on delivery of micro-dystrophin and micro-utrophin genes with recombinant adeno-associated viral vectors, exon skipping of the mutated dystrophin pre-mRNAs, reading through termination codons with small molecules that mask premature stop codons, dystrophin gene repair by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated genetic engineering, and increasing utrophin levels. Our analyses highlight the importance of various dystrophin and utrophin domains in Duchenne muscular dystrophy treatment, providing insights into designing novel therapeutic compounds with improved efficacy and decreased immunoreactivity. While the necessary actin and β-dystroglycan binding sites are present in both proteins, important functional distinctions can be identified in these domains and some other parts of truncated dystrophins might need redesigning due to their potentially immunogenic qualities. Alternatively, therapies based on utrophins might provide a safer and more effective approach.

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Section: Gene Editing With Crispr/cas9mentioning

confidence: 99%

Dystrophin- and Utrophin-Based Therapeutic Approaches for Treatment of Duchenne Muscular Dystrophy: A Comparative Review

Szwec,

Kapłucha,

Chamberlain

et al. 2023

BioDrugs

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Therapeutic approaches for Duchenne muscular dystrophy

Roberts,

Wood,

Davies

2023

Nat Rev Drug Discov

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Myospreader improves gene editing in skeletal muscle by myonuclear propagation

Poukalov,

Valero,

Muscato

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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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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Non-uniform dystrophin re-expression after CRISPR-mediated exon excision in the dystrophin/utrophin double-knockout mouse model of DMD

Cited by 6 publications

References 86 publications

Dystrophin- and Utrophin-Based Therapeutic Approaches for Treatment of Duchenne Muscular Dystrophy: A Comparative Review

Dystrophin- and Utrophin-Based Therapeutic Approaches for Treatment of Duchenne Muscular Dystrophy: A Comparative Review

Therapeutic approaches for Duchenne muscular dystrophy

Myospreader improves gene editing in skeletal muscle by myonuclear propagation

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