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”.

Gene Editing Does not Increase Mutation Rate in Stem Cells


Substituting one gene for another in cultured cells was once the stuff of science fiction, but with the ability to grow cells from our own bodies in culture and even convert them into embryonic-like stem cells, gene replacement has moved from the realms of science fiction to reality. However, the introduction of any all new technology comes with risks and trade-offs. In the case of gene replacement, there is the promise of fixing genes with mutations in them that cause genetic diseases. Unfortunately, any manipulation of the human genome runs the risk of adding new mutations to the genome whose side effects are unknown. Thus the cure might end up being worse than the disease itself.

New work from scientists at the Salk Institute in La Jolla, California has shown that new gene replacement techniques in stem cells does not increase the overall occurrence of mutations in those cultured cells. These new results were published the July 3 edition of the journal Cell Stem Cell.

“The ability to precisely modify the DNA of stem cells has greatly accelerated research on human diseases and cell therapy,” says senior author Juan Carlos Izpisua Belmonte, professor in Salk’s Gene Expression Laboratory. “To successfully translate this technology into the clinic, we first need to scrutinize the safety of these modified stem cells, such as their genome stability and mutational load.”

Introducing new genes into cells can occur by one of two methods. Engineered viruses can deliver new genes to a cell, which is then integrates the new DNA sequence in place of the old one. Alternatively, scientists can use custom targeted nucleases, such as TALEN proteins, which cut DNA at any desired location. Such proteins will extirpate the gene that needs to be replaced and then the new (potentially improved version of the gene) is simply added to the mix. The cell’s natural repair mechanisms will paste the new gene in place.

Belmonte’s lab has pioneered the use of modified viruses known as helper-dependent adenoviral vectors (HDAdVs) to fix genetic mutations that cause sickle-cell anemia. Sickle cell anemia is one of the most severe blood diseases found in the world. Belmonte and his collaborators have used HDAdVs to replace the mutant version of the globin gene in a stem cell line with a mutant-free version. This generated stem cells that could be theoretically be infused into patients’ bone marrow where they would create healthy blood cells.

Before such technologies are applied to humans, though, researchers must ascertain the risks of editing genes in stem cells. Even though both common gene-editing techniques have been shown to be accurate at altering the right stretch of DNA, concerns remain that the editing process could make the cells more unstable and prone to mutations in unrelated genes.

“As cells are being reprogrammed into stem cells, they tend to accumulate many mutations,” says Mo Li, a postdoctoral fellow in Belmonte’s lab and an author of the new paper. “So people naturally worry that any process you perform with these cells in vitro—including gene editing—might generate even more mutations.”

To test the safety of gene editing techniques, Belmonte’s research group, collaborated with BGI and the Institute of Biophysics, Chinese Academy of Sciences in China. They originally used a stem cell line that contains mutations in the beta-globin gene, which cause sickle-cell anemia. Belmonte then used HDAdV to edit the beta-globin genes of some cells, and edited the beta-globin genes of other cells by means of one of two TALEN proteins. Other cells were grown without any gene editing. Then, with the help of their Chinese collaborators, they fully sequenced the entire genome of each cell from the four edits and control experiment.

While all of the cells gained a low level of random gene mutations during the experiments, the cells that had undergone gene-editing—whether through HDAdV—or TALEN-based approaches—had no more mutations than the cells kept in culture.

“We were pleasantly surprised by the results,” Keiichiro Suzuki, a postdoctoral fellow in Belmonte’s lab and an author of the study, says. “People have found thousands of mutations introduced during iPSC reprogramming. We found less than a hundred single nucleotide variants in all cases.”

According to Li, this does not necessarily mean that there are no inherent risks to using stem cells with edited genes. However, it does mean that the editing process does not make stem cells that have undergone gene replacement are any less safe.

“We concluded that the risk of mutation isn’t inherently connected to gene editing,” he says. “These cells present the same risks as using any other cells manipulated for cell or gene therapy.” He adds that two other papers published in the same issue support their results (one by Johns Hopkins University and one from Harvard University and collaborators).

The next step for the Belmonte group is to determine if gene-repair in other cell types might be more likely to increase the mutation rate or if targeting other genes can cause unwanted mutations. They also hope that their findings will encourage those in the field to keep pursuing gene-editing techniques as a potential way to treat genetic diseases in the future.