CRISPR-inhibition for FSHD

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SUMMARY Facioscapulohumeral muscular dystrophy (FSHD) is a complex genetic and epigenetic disease caused by chromatin relaxation of the D4Z4 macrosatellite repeat array at chromosome 4q35, which leads to aberrant and pathogenic expression of the DUX4 gene in skeletal muscle. The most direct path to an FSHD therapy is eliminating expression of DUX4 mRNA. Importantly, data from clinically affected and asymptomatic FSHD subjects support that any reduction in DUX4 expression will have therapeutic benefit. CRISPR/Cas9 technology has been used extensively to modify specific genomic regions, offering the potential for permanent correction of many diseases. While the dangers associated with standard CRISPR editing are of particular concern in a repetitive region such as the FSHD locus, the use of CRISPR to repress gene expression is ideally suited to FSHD. We have shown that CRISPR inhibition (CRISPRi) consisting of dead Cas9 fused to a small transcriptional inhibitor can repress expression of DUX4 in FSHD myocytes, providing proof-of- principle that the pathogenic repeat can be successfully targeted and repressed. However, an effective therapy will require both efficient delivery of therapeutic components to skeletal muscles and long-term repression of the disease locus. To address these needs, we re- engineered our CRISPRi platform to allow in vivo delivery of larger and more powerful epigenetic repressors. We designed an FSHD-optimized regulatory cassette to drive the smaller dCas9 ortholog from S. aureus (dSaCas9) fused to four different epigenetic repressors (HP1, HP1, the MeCP2 transcriptional repression domain, or the SUV39H1 SET domain) that were previously too large to fit into AAV vectors. Targeting these factors to the DUX4 promoter or exon 1 returns the chromatin at the FSHD locus to a more normal state of repression, reducing expression of DUX4 and its target genes with no deleterious effects on the muscle transcriptome. In this proposal, we will undertake the first in vivo assessment of a CRISPR- based approach to treating FSHD, using new vectors in which all CRISPRi components are contained within single therapeutic cassettes. These will be tested in cultured primary FSHD myocytes and in two FSHD mouse models that provide complementary advantages for pre- clinical assessment. The premise of this proposal is that AAV-mediated dCas9 targeting of epigenetic repressors to DUX4 can effectively and stably silence the disease locus without deleterious off-target effects. Successful completion of this project will provide the preclinical validation for a treatment that corrects the fundamental epigenetic dysregulation in FSHD.