#apaperaday: Correction of exon 2, exon 2–9 and exons 8–9 duplications in DMD patient myogenic cells by a single CRISPR/Cas9 system

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: Correction of exon 2, exon 2–9 and exons 8–9 duplications in DMD patient myogenic cells by a single CRISPR/Cas9 system

Today’s pick is on gene editing of  cultured muscle cells from 3 Duchenne patients with different duplication mutations by Lemoine et al published in @SciReports DOI: 10.1038/s41598-024-70075-5.

Gene editing (CRISPR/Cas9) allows reading frame restoration on DNA level rather than RNA level (where antisense oligonucleotides, ASOs act). RNA is shortlived, so ASO treatment needs to be repeated. Gene editing would be a 1 time treatment, IF delivery & safety issues are solved.

Duplications of one or more exons occur in ~10% of Duchenne patients. ASOs can only be used for single exon duplications. For other duplications, things are challenging as you want the ASO to target 1 of the duplicated exons but not the other. So this diluted efficiency.

Gene editing by contrast is easier for duplications, because you need only 1 guideRNA to remove the duplicated area (it will cut twice, but as there is a duplication, the target is the same). Furthermore, after cutting a full length dystrophin is formed.

Other researchers have shown gene editing for duplication is feasible. Here authors expanded the repertoire by editing an exon 2,  exon 2-9 and and exon 8-9 duplication. They obtained immortalized myoblasts from each mutation and verified the duplication and absence of dystrophin

Then they mapped the breakpoints so they could optimally design the guides, to remove as much as possible from the duplication, without affecting normal splicing. Multiple guides were designed and efficiency was confirmed in a test system.

Then the most optimal guides and Cas9 were transfected into the patient cells (respective guides in respective cells obviously). Authors isolated clones, and expanded those. They had several clones for each mutation where the editing had been successful.

These clones were differentiated and it was shown they could make dystrophin by Western blot & immunofluorescence. Authors also performed RNAseq but the challenge was that they had only 1 wild type cell and there was more difference between cell lines than corrected/uncorrected.

This is not surprising as edited cells have the same genetic background as mutated cells, while the wild type had a different genetic back ground. This did make it difficult to study treatment effects however. Authors looked into genes up or down regulated in each patient cell

These genes changed after treatment, however, not always in the direction of wild type. This is a challenge & it highlights differences between cell lines of different genetic background and the careful selection and study design needed when using transcriptomics or proteomics.

Authors further discuss that even though they had clones that were corrected (so only corrected cells), which should make wild type levels of dystrophin, they saw only 20% of dystrophin. They argue perhaps there are epigenetic changes that prevent higher expression.

This needs to be studied in more detail. Fully agree and I appreciate the authors flagging this point as the chromatin change (if it exists) would hamper dystrophin restoration with gene editing and exon skipping both. Finally authors outline the study so far is only in vitro.

They are generating a mouse model to study things further in vivo. Looking forward to more results – also (especially) about the chromatin changes or other causes of lower dystrophin expression in corrected cells. The dystrophin gene never ceases to intrigue…