Gene Editing in iPS Cells Corrects Genetic Mutations That Cause Muscular Dystrophy

Induced pluripotent stem cells or iPSCs have many of the same characteristics as embryonic stem cells. One such feature is the ability to be grown in culture and manipulated like genuine tissue culture cells.

To that end a research group at the Center for iPS Cell Research and Application (CiRA) have used iPSCs made from the cells of patients with Duchenne muscular dystrophy (DMD) to show that such mutations can be efficiently fixed.

This research, which was published in Stem Cell Reports, demonstrates how a new group of engineered nucleases, such as TALEN and CRISPR, can edit the genome of iPS cells generated from skin cells isolated from a DMD patient. After being genetically fixed, these iPSCs were differentiated into skeletal muscles, and it was clear that the mutation responsible for DMD had disappeared.

DMD is a severe muscular degenerative disease caused by loss-of-function mutations in the dystrophin gene. DMD affects 1 in 3500 boys and normally leads to death by early adulthood. The treatments for this disease are largely palliative.

However, the capability to edit the genomes of mutant cells is a formerly unknown option that was once only for the realms of science fiction. Two nucleated called TALEN and CRISPR have quickly become invaluable tools in molecular biology. These enzymes allow scientists to cleave genes at specific locations and then modify the cut ends to generate a specifically chosen genomic sequence. However, these programmable nucleases are not perfect and often mistakenly edit similar sequences that vary a few base pairs from the target sequence. This makes them unreliable for clinical use because of the potential for creating new, undesired mutations.

For precisely this reason, iPSCs are ideal model systems because they provide researchers an abundance of patient cells on which to test the programmable nucleated, and determine the optimal conditions that minimize off-target modifications. CiRA scientists used this very feature to generating iPS cells from a DMD patient. Then they utilized several different TALENs and CRISPRs to modify the genome of the iPS cells, which were then differentiated into skeletal muscle cells. In all cases, dystrophin protein expression was restored, and in some cases, the dystrophin gene was fully corrected.

One of the reasons for the success in this project was the development of a computational protocol that minimized the risk of off-target editing. The CiRA team built a database that contained all possible combination of sequences up to 16 base pairs long. Among these, they isolated those sequences that only appear once in the human genome. DMD can be caused by several different mutations. For example, in the case of the patient used in this study, it was the result of the deletion of exon 44. After building a histogram of unique sequences that appeared in a genomic region that contained this exon, the CiRA group found a cluster of unique sequences in exon 45.

The head researcher for this project, Akitsu Hotta, who headed the project and holds joint positions at CiRA and the Institute for Integrated Cell-Materials Sciences at Kyoto University, said:  “Nearly half the human genome consists of repeated sequences. So even if we found one unique sequence, a change of one or two base pairs may result in these other repeated sequences, which risks the TALEN or CRISPR editing an incorrect region. To avoid this problem, we sought a region that hit high in the histogram.”

This paper provides a proof-of-principle for using iPS cell technology to treat DMD in combination with TALEN or CRISPR. The group now aims to expand this protocol to other diseases.  First author Lisa Li explains, “We show that TALEN and CRISPR can be used to correct the mutation of the DMD gene. I want to apply the nucleases to correct mutations for other genetic-based diseases like point mutations”.