misshapen nuclear envelopes
(red) from iPSCs (DNA in blue).
The right panel shows
gene-edited iPSCs.
Credit: Salk Institute
Results of new research may ease previous concerns that gene-editing techniques could add unwanted mutations to stem cells.
Researchers compared gene editing techniques in lines of induced pluripotent stem cells (iPSCs) derived from a patient with sickle cell disease (SCD).
And they found that neither viral nor nuclease-based gene-editing methods increased the frequency of mutations in the iPSCs.
The team reported these results in Cell Stem Cell.
“The ability to precisely modify the DNA of stem cells has greatly accelerated research on human diseases and cell therapy,” said senior study author Juan Carlos Izpisua Belmonte, PhD, of the Salk Institute for Biological Studies in La Jolla, California.
“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.”
Previously, Dr Belmonte’s lab pioneered the use of modified viruses, called helper-dependent adenoviral vectors (HDAdVs), to correct the genetic mutation that causes SCD.
He and his colleagues used HDAdVs to replace the mutated gene in a line of iPSCs with a mutant-free version, creating stem cells that could, theoretically, be infused into patients’ bone marrow and help create healthy blood cells.
Before such technologies are applied to humans, though, Dr Belmonte and his colleagues wanted to know whether there were risks related to editing the genes in iPSCs.
“As cells are being reprogrammed into stem cells, they tend to accumulate many mutations,” said Mo Li, PhD, a postdoctoral fellow in Dr Belmonte’s lab.
“So people naturally worry that any process you perform with these cells in vitro—including gene editing—might generate even more mutations.”
To find out whether this was the case, the researchers conducted tests in a line of SCD-derived iPSCs.
They edited the genes of some cells using 1 of 2 HDAdV designs. And they edited others using 1 of 2 transcription activator-like effector nuclease (TALEN) proteins.
They kept the rest of the SCD iPSCs in culture without editing them. Then, the team sequenced the entire genome of each cell from the 4 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,” said Keiichiro Suzuki, PhD, a postdoctoral fellow in Dr Belmonte’s lab.
“People have found thousands of mutations introduced during iPSC reprogramming. We found less than a hundred single nucleotide variants in all cases.”
The researchers noted that this finding doesn’t necessarily mean there are no inherent risks to using stem cells with edited genes. However, it does suggest the editing process doesn’t make iPSCs any less safe.
“We concluded that the risk of mutation isn’t inherently connected to gene editing,” Dr Li said. “These cells present the same risks as using any other cells manipulated for cell or gene therapy.”
The Belmonte group is now planning more studies to address whether gene-repair in other cell types, using other approaches, or targeting other genes could be more or less likely to cause unwanted mutations.
For now, they hope their findings encourage those in the field to keep pursuing gene-editing techniques as a potential way to treat genetic diseases in the future.
