Gene editing can correct mutations causing Duchenne muscular dystrophy in human cells and mice
Duchenne muscular dystrophy (DMD) is a rare disease linked to mutations in the Dystrophin gene on the X chromosome. This can occur in various forms, but disrupts the correct readthrough of the protein's sequence - resulting in a defective protein and severe muscle atrophy.
There are a number of research teams working on treatments for muscular dystrophy in its various forms, including gene therapy and pharmaceutical approaches, but perhaps the most elegant treatment would be to correct the mutation using a platform such as CRISPR. CRISPR-Cas9 is the most famous version of the renowned CRISPR technology, but a new variant CRISPR-Cpf1 offers some advantages - snipping only one strand of DNA rather than two. CRISPR itself can be thought of as a guiding sequence, which homes in on a specific sequence of DNA. Attaching an enzyme called an endonuclease (which snips DNA) enables customisation - allowing you to directly edit a sequence or silence it.
A group of researchers from the University of Texas (UT) Southwestern Medical Center used CRISPR-Cpf1 in human induced pluripotent stem cells derived from fibroblast cells. They were able to correct mutations which disrupted dystrophin mRNA - restoring an almost full length, and functional protein. When they differentiated these cells into cardiomyocytes types, the cells showed similar activity to normal health ones.
“The authors use CRISPR-Cfp1 to correct the Dmd mutation in vitro and ex vivo.This study nicely adds to the repertoire of tools available for Dmd editing, showing that Cpf1—like Cas9 nucleases evaluated in prior studies—is also effective for targeting frame-disrupting mutations in Dmd. All the published experimental gene repair therapy for DMD relies on making two cuts surrounding the mutated exon to remove the mutation. The researchers here used a single guide RNA so the genome only needs to be cut once, which increases efficiency”
The scientists next tested the strategy on mouse zygotes before implantation in a surrogate mother. 5 of 24 treated mice zygotes demonstrated correction of the mutation. The method therefore seems highly promising, but there are big challenges remaining towards translation. There are hurdles when it comes to delivering the platform into enough human cells in adults or children. However, the study again underlines how effective gene editing techniques could be, once properly developed and optimised.
“The dystrophin editing with Cas9 and Cfp1 appears to have similarly high efficiencies without apparent off-target effects. If the percentage of zygotes with the corrected allele equates to the number of cells within a tissue that is properly corrected, that 20 percent efficiency is relatively high, and would likely restore function to the tissue. There is concern about the immune response to these nucleases. We’re not sure yet whether one of these nucleases is better tolerated or which will work better in patients. That’s why we’re working on both”
Read more at The Scientist