Proof of concept for new strategy of genome editing to correct splice-site mutations


Most human genes contain exons that are translated into protein and introns that are removed during the translation from RNA to protein. The process of removing introns and stitching axons together is called splicing. While mutations in introns are not translated into protein, they may interfere with the spicing process. This can result in proteins with missing or altered exons. Splice-site mutations underlie diseases such as β-thalassemia, epilepsy and muscular dystrophy.


A study that was published in Nature Medicine this week tried to treat the latter disease with a new CRISPR strategy. In the dy2J/dy2J mouse model of congenital muscular dystrophy 1, the team from The Hospital for Sick Children Research Institute in Toronto, Canada made 2 double strand breaks around the mutation in the second intron. For repair, the researchers relied on non-homologous end joining, a DNA damage repair pathway that does not rely on a template. This is in contrast to the homologous recombination pathway that most CRISPR genome editing studies so far have used. To achieve homologous recombination, a therapy has to provide a corrective template as well as the CRISPR tools to each cell. While the non-homologous end joining strategy is limited to applications where part of the sequence can be removed rather than has to be corrected, it is more efficient as there are fewer steps in the process. Furthermore, homologous recombination is know to be inefficient some tissues, including muscles. 


In the mouse model, the researchers achieved ~25% healthy, full length protein. This was associated with reduced fibrosis, increased muscle mass and improved muscle architecture compared to control mice. Force generated by the muscles was restored to the healthy, wild-type range. The mice were either treated at two days old with a systemically administration or with an intramuscular injection at 3 weeks old and followed until 10 weeks of age. This implies that the treatment needs to be administered before onset of symptoms or as soon as possible thereafter, although this will need to be examined in more detail. Further safety studies investigating potential off-target cuts or inappropriate repair of the cuts will also need to be conducted. The study is an interesting proof of concept for the treatment of diseases where part of the gene can be removed, rather than repaired and is particularly interesting for targeting tissues in which homologous repair is know to be inefficient. 


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