FP.29 AAV-CRISPR-Cas13 gene therapy for FSHD: DUX4 gene silencing efficacy and immune responses to Cas13b protein

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is among the most prevalent muscular dystrophies, ranging from 1 in 8,333 to 1 in 20,000. Currently no treatment exists that alters the course of FSHD, and therapy development remains an unmet need in the field. Abnormal reactivation of the <i>DUX4</i> gene in skeletal muscle has emerged as an underlying cause of muscle weakness and wasting in FSHD. We propose that <i>DUX4</i> silencing is the most direct route to FSHD therapy. Toward this goal, we developed an AAV6-CRISPR-Cas13 strategy to silence <i>DUX4</i> mRNA. Cas13 targets and cleaves RNA instead of DNA, and avoids potential risks of permanent off-target genome editing that could arise with DNA-targeting systems. Intramuscular delivery of an AAV6 vector encoding a PspCas13b enzyme and <i>DUX4</i>-targeting guide RNAs reduced <i>DUX4</i> mRNA by >50% and improved histopathological outcomes in FSHD mice. To investigate possible off-target effects, we performed RNA-seq of treated versus control or untreated human myoblasts and also examined potential collateral RNA cleavage activity using a dual reporter system. Although we did not detect collateral cleavage, our RNA-sequencing results suggested some guide RNAs could induce potential off-target gene expression changes. We are currently exploring mechanisms to explain these differential off-target effects. To address whether PspCas13b can activate a mammalian host immune response, we injected wild-type mice with AAV-Cas13b and investigated immune cell infiltration and pro-inflammatory cytokine profiles. We find evidence of an immune response against PspCas13b in injected mouse muscles. Importantly, transient immunosuppression reduced immune responses to Cas13b in treated animals. In conclusion, our data support that Cas13b can target and reduce <i>DUX4</i> expression in FSHD muscles, but minimizing cellular immune response may be necessary to translate AAV-Cas13b therapy.