I. Luksch(1,2), T. Bozoglu(1,2), T. Ziegler(1,2), C. M. Poch(1,2), N. Raad(1), M. Stirm(3), A. Bähr(1,2), N. Klymiuk(1,2), K. L. Laugwitz(1,2), A. Moretti(1,2), E. Wolf(3), J. Grünewald(1,2), C. Kupatt(1,2)

1. Klinik und Poliklinik für Innere Medizin I, University Clinic Rechts der Isar, School of Medicine and Health, Technical University of Munich; 2. DZHK (German Center for Cardiovascular Research), partner site Munich Heart Alliance, Munich, Germany; 3. Chair for Molecular Animal Breeding and Biotechnology, Gene Center and Department of Veterinary Sciences, and Center for Innovative Medical Models (CiMM), LMU Munich

Duchenne Muscular Dystrophy (DMD) is the most frequent hereditary childhood myopathy leading to progressive muscle degeneration and, ultimately, premature death due to respiratory and cardiac failure. It’s caused by the absence of Dystrophin, usually resulting from frameshift mutations in the 427kDa-protein encoding gene.

In this study, we aim to achieve exon skipping by developing a base editor (BE) delivered as dual-AAV-system targeting the splice acceptor site (SAS) of exon 53 with the benefit of avoiding double strand breaks.

Screening of various deaminase and Cas9 combinations, together with suitable sgRNAs was performed in primary kidney fibroblasts of a DMDΔ52-pig. Finally, editing efficacies up to 17% could be observed using a dual-AAV-system containing the ABE8e-BE and spRY-Cas9 targeting the SAS of exon 53. The two most promising dual-AAV-constructs were packed as AAVs and used to transduce ex vivo heart slices, followed by extensive analysis. Furthermore, AAVs containing orthologous spacers were applied to human iPSC-derived DMDΔ52 cardiomyocytes resulting in an editing efficacy of 9.2% and an exon skip rate of 10.4% in 2D culture. In 3D engineered heart patches, an editing efficacy of 2.1% could be achieved, resulting in a reduction of the arrhythmic load and normalization of the effective refractory period.

Finally, in vivo application of the AAVs intrathoracicly to hDMDΔ52/mdx-mice and intracoronarily to DMDΔ52-pigs is scheduled, followed by electrophysiological, molecular and histological analyses.

This study aims to demonstrate that base editing may critically improve efficacy and safety of gene editing in DMD, a rapidly progressing disease with few effective alternate options.