Written by: Stephen Hsu
Primary Source: Information Processing
Safe and Effective soon for humans?
Science DOI: 10.1126/science.aad5143
Duchenne muscular dystrophy (DMD) is a devastating disease affecting about 1 out of 5000 male births and caused by mutations in the dystrophin gene. Genome editing has the potential to restore expression of a modified dystrophin gene from the native locus to modulate disease progression. In this study, adeno-associated virus was used to deliver the CRISPR/Cas9 system to the mdx mouse model of DMD to remove the mutated exon 23 from the dystrophin gene. This includes local and systemic delivery to adult mice and systemic delivery to neonatal mice. Exon 23 deletion by CRISPR/Cas9 resulted in expression of the modified dystrophin gene, partial recovery of functional dystrophin protein in skeletal myofibers and cardiac muscle, improvement of muscle biochemistry, and significant enhancement of muscle force. This work establishes CRISPR/Cas9-based genome editing as a potential therapy to treat DMD.
It seems the efficiency of the treatment is sufficient to produce measurable improvement of the condition, although the deletion is only effected in a small fraction of cells / DNA. How does the editing proceed in time? Does the virus vector completely die out in the tissue after some period of time?
… The Cas9 and gRNA AAV vectors were premixed in equal amounts and injected into the tibialis anterior muscle of mdx mice. Contralateral limbs received saline injection. At eight weeks post-injection, the muscles were harvested and analyzed for deletion of exon 23 from the genomic DNA and mRNA, and expression of dystrophin protein. End-point PCR across the genomic locus revealed the expected ~1,171 bp deletion in all injected limbs (Fig. 1b). Droplet digital PCR (ddPCR) was used to quantify the percent of modified alleles by separately amplifying the unmodified or deleted DNA templates. ddPCR showed that exon 23 was deleted in ~2% of all alleles from the whole muscle lysate (Fig. 1c). Sanger sequencing of gel-extracted bands confirmed the deletion of exon 23 as predicted without any additional indels (Fig. 1b).
… Next, we assessed muscle function. The specific twitch (Pt) and tetanic (Po) force were significantly improved in Cas9/gRNA-treated muscle. … Collectively these results show that CRISPR/Cas9-mediated dystrophin restoration improved muscle structure and function.
… More broadly, this work establishes CRISPR/Cas9-mediated genome editing as an effective tool for gene modification in skeletal and cardiac muscle and as a therapeutic approach to correct protein deficiencies in neuromuscular disorders and potentially many other diseases. The continued developed of this technology to characterize and enhance the safety and efficacy of gene editing will help to realize its promise for treating genetic disease.