Targeted epigenetic repression by CRISPR/dSaCas9 suppresses pathogenic DUX4-fl expression in FSHD
Charis L Himeda1, Takako I Jones1, Peter L Jones1
1Department of Pharmacology, University of Nevada, Reno School of Medicine, Reno, NV 89557, USA.
Abstract:
Facioscapulohumeral muscular dystrophy (FSHD) is caused by incomplete silencing of the disease locus, leading to pathogenic misexpression of DUX4 in skeletal muscle. Previously, we showed that CRISPR inhibition could successfully target and repress DUX4 in FSHD myocytes. However, an effective therapy will require both efficient delivery of therapeutic components to skeletal muscles and long-term repression of the disease locus. Thus, we re-engineered our platform to allow in vivo delivery of more potent epigenetic repressors. We designed an FSHD-optimized regulatory cassette to drive skeletal muscle-specific expression of dCas9 from Staphylococcus aureus fused to HP1α, HP1γ, the MeCP2 transcriptional repression domain, or the SUV39H1 SET domain. Targeting each regulator to the DUX4 promoter/exon 1 increased chromatin repression at the locus, specifically suppressing DUX4 and its target genes in FSHD myocytes and in a mouse model of the disease. Importantly, minimizing the regulatory cassette and using the smaller Cas9 ortholog allowed our therapeutic cassettes to be effectively packaged into adeno-associated virus (AAV) vectors for in vivo delivery. By engineering a muscle-specific epigenetic CRISPR platform compatible with AAV vectors for gene therapy, we have laid the groundwork for clinical use of dCas9-based chromatin effectors in skeletal muscle disorders.
Insights
This study developed a novel CRISPR-based gene therapy for facioscapulohumeral muscular dystrophy (FSHD). The approach uses epigenetic repressors delivered via AAV vectors to silence the DUX4 gene in skeletal muscles, offering a promising therapeutic strategy.
Area of Science:
- Genetics
- Molecular Biology
- Gene Therapy
Background:
- Facioscapulohumeral muscular dystrophy (FSHD) results from aberrant DUX4 gene expression in skeletal muscle.
- Previous CRISPR inhibition showed promise but lacked efficient in vivo delivery and long-term repression.
- Developing effective therapies requires targeting the DUX4 locus for sustained gene silencing.
Purpose of the Study:
- To engineer an improved CRISPR-based epigenetic repression platform for FSHD.
- To achieve efficient in vivo delivery of therapeutic components to skeletal muscles.
- To establish long-term repression of the DUX4 disease locus.
Main Methods:
- Designed an FSHD-optimized regulatory cassette for muscle-specific expression of dCas9 fused to epigenetic repressors (HP1α, HP1γ, MeCP2, SUV39H1).
- Targeted the dCas9-epigenetic repressor complex to the DUX4 promoter/exon 1.
- Utilized adeno-associated virus (AAV) vectors for in vivo delivery of minimized therapeutic cassettes.
Main Results:
- Targeting repressors to the DUX4 locus increased chromatin repression.
- DUX4 and its target gene expression were suppressed in FSHD myocytes and a disease mouse model.
- Minimized cassettes and smaller Cas9 orthologs enabled effective AAV packaging for in vivo application.
Conclusions:
- Developed a muscle-specific epigenetic CRISPR platform compatible with AAV vectors.
- Demonstrated successful DUX4 repression in preclinical models of FSHD.
- Laid the groundwork for clinical translation of dCas9-based gene therapy for skeletal muscle disorders like FSHD.
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