Open Scientific Literature

Background: Duchenne muscular dystrophy (DMD) is a severe, progressive neuromuscular disease with onset in early childhood, making the development of effective therapies a critically important task, particularly in pediatrics. One of the promising approaches is genome editing using the CRISPR/Cas9 system, aimed at restoring the reading frame of the mutated DMD gene. Aim: In vitro evaluation of the efficacy of two CRISPR/Cas9 editing strategies for DMD therapy: 1) induction of single double-strand breaks in splicing sites to skip exons 43-55, and 2) induction of two breaks in introns to completely remove exons 11 and 12 in order to restore the reading frame. Methods: An experimental in vitro study was conducted. The targets selected were donor/acceptor splicing sites of exons 43-55 of the DMD gene (for strategy 1) and sequences in introns 10 and 12 (for strategy 2). For SpCas9 and SaCas9 nucleases, guide RNAs (gRNAs) were selected via in silico screening. The constructs were cloned into plasmid vectors. Primary gRNA screening was performed on the HEK293T cell line. Validation was carried out on immortalized myoblasts from a healthy donor (AB1190) and patient-specific myoblasts with a deletion of exons 48-54 (P1789). The primary endpoints were: 1) editing efficiency at the genomic DNA level, assessed by the frequency of indels using sequencing (TIDE, DECODR, NGS); 2) exon skipping efficiency at the mRNA level, assessed by targeted NGS of cDNA. Results: For the first strategy, 6 highly effective gRNAs were selected. Their editing activity at the DNA level in AB1190 myoblasts ranged from 2.8% to 46.05%. However, neither in AB1190 myoblasts nor in patient-specific P1789 myoblasts did these gRNAs lead to detectable skipping of the target exons (45, 50, 52, 53, 54, 55) at the mRNA level. For the second strategy, two pairs of gRNAs induced extensive deletions removing exons 11 and 12. The efficiency of the target deletion formation at the DNA level for the SpCas9-based p