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.

Stem Cells to Make Red Blood Cells and Platelets in Culture

A collaborative study between Boston University School of Public Health and researchers at Boston Medical Center has used induced pluripotent stem cells to make unlimited numbers of human red blood cells and platelets in culture.

This finding could potentially reduce the need for blood donations to treat patients who require blood transfusions. Such research could also help researchers examine fresh and new therapeutic targets in order to treat blood diseases such a sickle-cell anemia.

The lead scientist on this project was George Murphy, assistant professor of medicine at Boston University School of Medicine and co-director of the Center for Regenerative Medicine at Boston University. Murphy’s main collaborator was David Sherr, professor of environmental health at Boston University School of Medicine and the Boston University School of Public Health.

Induced pluripotent stem cells or iPSCs are made from adult cells by applying genetic engineering technology to the adult cells that introduces genes into them. The introduction of four specific genes de-differentiates the adult cells into pluripotent stem cells that can, potentially, differentiate into any adult cell type. This makes iPSCs powerful tools for research and potential therapeutic agents for regenerative medicine.

In this study, Murphy and others used iPSCs from the CreM iPS Cell Bank and exposed them to a battery of different growth factors in order to push them to differentiate into different adult cell types. They were looking for the precise cocktail to differentiate iPSCs into red blood cells, since they wanted to further study red blood cell development in detail.

One group of compounds given to the set of iPSCs were molecules that activate “aryl hydrocarbon” receptors. Aryl hydrocarbon receptors (AHRs) play important roles in the expansion of hematopoietic stem cells, which make blood cells, since antagonism of AHRs promotes expansion of hematopoietic stem cells (see AE Boitano et al.,Science 10 September 2010: Vol. 329 no. 5997 pp. 1345-1348). In this case, however, Murphy and his colleagues observed a dramatic increase in the production of functional red blood cells and platelets in a short period of time. THis suggests that the ARH is important for normal blood cell development.

Aryl Hydrocarbon Receptor
Aryl Hydrocarbon Receptor

“This finding has enabled us to overcome a major hurdle in terms of being able to produce enough of these cells to have a potential therapeutic impact both in the lab and, down the line, in patients,” said Murphy. “Additionally, our work suggests that AHR has a very important biological function in how blood cells form in the body.”

“Patient-specific red blood cells and platelets derived from iPSC cells, which would solve problems related to immunogenicity and contamination, could potentially be used therapeutically and decrease the anticipated shortage and the need for blood donation,” added Murphy.

iPS-derived cells have tremendous potential as model systems in which scientists can test and develop new treatments for disease, given that such diseases can be constructed in the laboratory. These iPSC-derived red blood cells could be used by malaria researchers, and IPSC-derived platelets could be used to explore cardiovascular disease and treatments for blood clotting disorders.

Because my mother died from myelodysplasia, this finding has some personal interest to me. Mom had a difficult blood type to match, since she had the Bombay blood type (H). Finding blood for her was a major tour de force, and as she received blood that was less and less well matched to her body, she suffered the ravages of poorly matched blood. A treatment of red blood cells made from IPSCs derived from her own cells might have extended her life and even improved her quality of life in her later years.

I look forward to this research eventually culminating in clinical trials.