All-in-one vectors for epigenetic CRISPR inhibition of DUX4-fl in facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by incomplete epigenetic silencing of the disease locus, leading to pathogenic misexpression of DUX4 in skeletal muscle. Previously, we showed that CRISPR inhibition (CRISPRi) using several epigenetic regulators successfully targets and represses DUX4 in FSHD myocytes with no adverse effects on the muscle transcriptome. However, for translatability, adeno-associated virus (AAV)-mediated gene therapies must include all components within a single vector that expresses at therapeutic levels in all target tissues. Thus, we have re-engineered our CRISPRi platform to an all-in-one (AIO) single-vector system. Key modifications were as follows: (1) optimizing our regulatory cassette for high activity in all skeletal muscles while maintaining low or no activity in FSHD-unaffected tissues and (2) minimizing epigenetic regulators to their essential functional repressor domains, together creating enough space to include a single guide RNA (sgRNA) expression cassette within the AAV packaging limit. Targeting these AIO cargos to DUX4 suppressed pathogenic gene expression in FSHD1 and FSHD2 myocytes. Importantly, AAV-mediated delivery of our best AIO cargo also repressed DUX4 in an FSHD xenograft mouse model. These therapeutic cassettes represent a clinically relevant CRISPRi platform for gene therapy of FSHD and a useful proof-of-concept platform for other skeletal muscle gene therapies.