The CRISPR-Cas9 method of gene correction has been shown to be effective in mice for treating Duchenne muscular dystrophy, bringing the gene editing technique closer to human trials.
One of the latest trials of the technique inserted a CRISPR-Cas9 edited gene delivered using an adeno-associated virus (AAV). After treatment, mice showed an improvement in both function and histology.
More than one in 5,000 newborn males suffer from Duchenne muscular dystrophy: The most common form of the disease. If the condition is left untreated, individuals develop severe muscular problems. Muscles weaken as a result of a mutation in the gene responsible for the 427-kDa cytoskeletal protein dystrophin. Loss of ambulation is typically seen by around 10-12 years of age. Both skeletal and cardiac muscles are affected, and sufferers of the condition can only expect a lifespan of around 25 years. By the time individuals reach their mid-twenties, the condition has severely affected cardiac muscles and patients usually die from heart failure.
Muscle strength can be improved with corticosteroids, although even with treatment, patients will only be expected to gain an extra 1-2 years of ambulation. Consequently, treatment of Duchenne muscular dystrophy has focused on correcting the molecular defect responsible. Eteplirsen has been used to skip exon 51 of the DPD gene, extending the period of ambulation by around 4 years and slowing the deterioration of the condition.
While gene replacement therapy is theoretically possible, due to the size of the DPD gene – it is too large to be delivered by an adeno-associated virus (AAV) – efforts to replace the faulty DMD gene have been hampered. However, the CRISPR-Cas9 method of gene editing has shown great promise. Three studies have now been performed that have targeted the mutation in exon 23 of the mdx mouse – mice which have a point mutation in the DMD gene that changes the amino acid coding for glutamine to threonine, resulting in the formation of non-functional dystrophin proteins.
All of the studies used a two-vector system to deliver AAV CRISPR instead of a single vector for delivering gRNA and the Cas9 enzyme. In these studies, the Cas9 from both Streptococcus pyogenes and Staphylococcus aureus were used and both were shown to be effective in pre-clinical in vivo studies on mdx mice. Both were also found to be safe and could therefore potentially be used in human trials.
Mice treated using this two-vector CRISPR-Cas9 technique showed a reduction in muscle necrosis, decreased fibrosis, and fewer infiltrating inflammatory cells. After treatment, mice also had increased grip strength, better resistance against eccentric contraction, improved force generation, and reduced serum creatine kinase. All three studies showed the restoration of dystrophin in cardiomyocytes.
While there are fears that CRISPR-Cas9 can result in off-target gene editing, no evidence was discovered of off-gene editing that was of clinical concern.
Now that the effectiveness of the technique has been shown in mdx mice, studies will now progress to humanized DMD mutations in animal models. Should the trials progress as planned, this technique could potentially be used to treat approximately 80% of individuals suffering from Duchenne muscular dystrophy.