Non-uniform dystrophin re-expression after CRISPR-mediated exon excision in the dystrophin/utrophin double-knockout mouse model of DMD

Abstract

Duchenne muscular dystrophy (DMD) is the most prevalent inherited myopathy affecting children, caused by genetic loss of the gene encoding the dystrophin protein. There are currently four FDA-approved drugs for DMD that aim to restore expression of dystrophin by exon skipping using splice switching oligonucleotides. While these therapies require lifelong repeat administration, recent advancements in gene editing technologies have raised the possibility of achieving 'permanent exon skipping', and thereby curing the disease with a single treatment. Here we have investigated the use of the Staphylococcus aureus CRISPR/Cas9 system and a double-cut strategy, delivered using a pair of AAV9 vectors, for dystrophin restoration in the severely-affected dystrophin/utrophin double knock-out (dKO) mouse. Single guide RNAs were designed to induce double-strand DNA breaks on either side of Dmd exon 23, such that the intervening exon 23 sequence is excised when the flanking intronic regions are joined via the non-homologous end joining repair pathway. Exon 23 deletion was confirmed at the DNA level by PCR and Sanger sequencing, and at the RNA level by RT-qPCR. Restoration of dystrophin protein expression was demonstrated by western blot and immunofluorescence staining in mice treated via either intraperitoneal or intravenous routes of delivery. Dystrophin restoration was most effective in the diaphragm, where a maximum of 5.7% of wild-type dystrophin expression was observed. CRISPR treatment was insufficient to extend lifespan in the dKO mouse, and dystrophin was expressed in a within-fiber patchy manner in skeletal muscle tissues. Further analysis revealed a plethora of non-productive DNA repair events, including AAV genome integration at the CRISPR cut sites. This study highlights potential challenges for the successful development of CRISPR therapies in the context of DMD.

Exon skipping induces uniform dystrophin rescue with dose-dependent restoration of serum miRNA biomarkers and muscle biophysical properties

“…Treatment of mdx mice with PPMO conjugates resulted in a within-fiber uniform pattern of sarcolemmal dystrophin expression across dose levels ( Figure 5 ), consistent with our previous results. 9 This is in contrast to our observations in CRISPR-Cas9-treated muscle (see related article), 42 whereby dystrophin was expressed in a patchy manner. These findings point to a potential advantage of antisense oligonucleotide-mediated exon skipping over gene editing.…”

Myospreader improves gene editing in skeletal muscle by myonuclear propagation

“…Multiple observations suggest that AAV gene therapies encoding nuclear-targeted cargoes are restricted to progenitor myonuclei following mRNA export, translation, and subsequent re-import of the encoded protein (13)(14)(15). Consistent with this, we observed that a limited number of myonuclei in WT C57BL/6 mice are functionally transduced by AAV packaged with a GFP transgene (Fig.…”

“…Similar to endogenous mRNAs, gene therapy mRNAs are transcribed in successfully transduced nuclei, then exported and dispersed throughout the myonuclear domain ( 16, 36 ). Soluble and structural proteins translated from therapeutic mRNAs can then travel further, the latter perhaps being more restricted ( 14, 37 ), but nuclear-targeted proteins are trapped close to their progenitor domains ( 19, 38 ). Large nuclear-targeted proteins then have no way to passively exit the nuclear pore complex and are degraded within the nucleus over time ( 39 ), similar to other nucleocytoplasmic shuttling proteins ( 40 ).…”

“…S1A and S1C). Critically, the inability of Cas9-based editors to propagate efficiently to non-transduced nuclei has led to surprisingly few myonuclei containing Cas9 protein ( 13 ) and is partly responsible for the sparse restoration of dystrophin in Dmd-edited myofibers ( 14, 15 ). While small cytoplasmic proteins diffuse well throughout the myofiber (Fig.…”