Duchenne muscular dystrophy (DMD), a monogenic disorder characterized by progressive muscle degeneration, is one of the first diseases being targeted for therapeutic genome editing using nuclease- based methods (CRISPR/ZFN/TALEN). However, safety and persistence remains a concern. Long-term (1 year) persistence and safety of a single intravenous administration of an adeno-associated virus (AAV) and CRISPR was reported in mdx mouse model recently [1]. They reported that ‘AAV-CRISPR is immunogenic when administered to adult mice’, which can be ‘avoided by treating neonatal mice’, and also warned about ‘unintended genome and transcript alterations’. Here, the integration of the Cas9 protein in the exact two locations in the DMD gene which has been edited has been shown based on the same sequencing data (Accid:PRJNA485509). Transcriptomic data also shows Cas9 being expressed. There is an important distinction between AAV and Cas9 integration - while AAV integration can be tolerated, Cas9 integration is a huge, and unacceptable, danger. While there are use cases where the nuclease can be sent as as protein, any gene-therapy application for DMD would require delivery using AAV and the nuclease in a plasmid. So, there is no possible alleviation for this in the future, unless we are willing to accept transgenic humans as a trade-off for curing DMD.
Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype