Progress Update: Base editing of DUX4 somatic polyadenylation signal

Report by Darina Šikrová
See grant Base editing of DUX4 somatic polyadenylation signal

Facioscapulohumeral muscular dystrophy is caused by undesired expression of the DUX4 gene in skeletal muscles. From earlier studies we know that DUX4 expression in skeletal muscle cells requires a specific DNA sequence – a polyadenylation signal – to yield a toxic DUX4 protein. The primary goal of this project was to test a new CRISPR/Cas9 tool (adenine base editor or ABE) for rapid and efficient disruption of this polyadenylation signal so that no stable DUX4 protein can be formed in skeletal muscle.

During the 2-year period we were able to set up and test different versions of the ABE tool and to create muscle cells which have the desired change in the polyadenylation signal using the best performing version of the ABE tool. As anticipated, disrupting the DUX4 polyadenylation signal leads to decreased DUX4 levels in the edited cells. We managed to do this in muscle cells from four independent FSHD donors with different underlying genetics. Encouraged by the result, we now focus on the translatability of this approach for possible future therapeutic intervention. We have started to develop tools for the delivery of the ABE tool into an FSHD mouse model so we can test the editing efficiency of the DUX4 polyadenylation signal in a relevant animal model.

Lastly, one of the concerns using editing technologies in clinical application is their potential off-target effect in which other sites in the genome are also affected by the ABE tool. We looked at high-ranking candidate off-target sites for undesired editing to occur and only detected low levels of editing at some sites. Compared to the targeted DUX4 polyadenylation signal, the off-target editing was minimal and the genomic sites did not seem to be of functional significance.