Enhanced Cytosine Base Editing for Duchenne Muscular Dystrophy

The study addresses the challenge of tRNA-specific adenosine deaminase (TadA) variants showing dual activity on cytosine and adenosine bases. Using TadA ortholog screening and multi-sequence alignment, the Chinese researchers developed aTdCBE, which lacks adenosine deaminase activity.

The novel CBE was tested for its ability to correct exon 55 splicing in a humanised mouse model of Duchenne muscular dystrophy (DMD) with a deletion in exon 54 of the DMD gene. The deletion results in a premature stop codon in exon 55 and leads to the production of a non-functional dystrophin protein. The objective was to restore dystrophin expression by skipping exon 55 through precise cytosine base editing at the exon 55 splice acceptor site (SAS).

Six weeks after in vivo delivery of aTdCBE, DNA base editing at the exon 55 splice acceptor site was over 40%, indicating effective editing of the DMD gene. Immunostaining showed that dystrophin-positive muscle fibres reached almost 99% of wild-type levels, while western blot analysis confirmed about 60% restoration of dystrophin expression.

RT-PCR further confirmed successful exon 55 skipping, restoring the correct reading frame of the DMD gene. This led to the production of functional dystrophin protein, demonstrating efficient gene editing and significant dystrophin restoration in the treated mice.

This work was conducted by researchers from HuidaGene Therapeutics and elsewhere in China, and it was published yesterday in Nature Communications.