Single-vector base editing rescues a nonsense variant in a humanized mouse model of Duchenne muscular dystrophy

Topic:

Pre-Clinical Research

Poster Number: LB444

Author(s):

Ryan Marks, MSc, The Hospital for Sick Children, Sina Fatehi, The Hospital for Sick Children, Enzhe Khazeeva, The Hospital for Sick Children, Emily Bell, The Hospital for Sick Children, Lijun Chi, PhD, The Hospital for Sick Children, Bei Yan, The Hospital for Sick Children, Emily Huynh, The Hospital for Sick Children, Matthew Rok, The Hospital for Sick Children, Paul Delgado Olguín, PhD, The Hospital for Sick Children, Ronald Cohn, MD, The Hospital for Sick Children, Evgueni Ivakine, PhD, The Hospital for Sick Children

Duchenne muscular dystrophy (DMD) is a pediatric neuromuscular disorder caused by perturbations of the DMD gene resulting in loss-of-function dystrophin, a structural protein located at the sarcolemma. Dystrophinopathy compromises the mechanical integrity of skeletal/cardiac myofibers, culminating in progressive muscle wasting and cardiorespiratory dysfunction often fatal by the third decade of life. Adenine base editors (ABEs) are CRISPR-derived technologies capable of performing programmable A•T-to-G•C transitions in situ via engineered ecTadA-mediated deoxyadenosine deamination, however translational applications have been limited due to target-adenine promiscuity (bystander edits), RNA off-targeting, and packaging constraints of clinically validated myotropic AAV vectors. Here, we developed the first therapeutic application of single AAV-compatible ABEs for precise correction of nonsense variants capable of restoring full-length dystrophin production and functional rescue in a humanized mouse model of DMD. We identified a patient harboring a de novo nonsense variant DMD c.9445C>T (p.Q3149X) amenable to ABE correction. Establishing this variant on our novel fully-humanized D2.mdx-hDMD9445C>T mouse model recapitulated histopathological hallmarks including loss of dystrophin production and laminin-α2 co-localization, severe endomysial thickening with fibrotic infiltrates, and centralized nuclei throughout cardiac and skeletal muscles, respectively. We show that monomeric TadA8e deaminase configurations could efficiently correct DMD9445C>T up to 88.2±1.7% in patient-derived primary myoblasts ex vivo when coupled with diverse, compact Cas9 nickase orthologues. Furthermore, we demonstrated that rational engineering of bespoke TadA8e deaminases, in combination with modified gRNA spacer and scaffold architectures, synergistically reduced bystander edits while eliminating off-target RNA editing with minimal compromise in therapeutic activity (49.9±7.4%). Notably, a single intravenous administration of 5E13vg•kg-1 MyoAAV4A-CK8e-ABE in 4-week-old D2.mdx-hDMD9445C>T mice achieved ~85% dystrophin rescue in the heart and 20-40% across multiple skeletal muscles. AAV-ABE treatment significantly improved cardiac function, grip strength, and overall muscle architecture as evaluated 3 months post-injection. These findings substantiate ABE as efficient tools for therapeutic genome editing and highlight precision and safety optimizations essential for clinical translation.