The ability to switch out one gene for another in a line of living stem cells has only crossed from science fiction to reality within this decade. As with any new technology, it brings with it both promise-the hope of fixing disease-causing genes in humans, for example-as well as questions and safety concerns.

Now, Salk scientists have put one of those concerns to rest: using gene-editing techniques on stem cells doesn't increase the overall occurrence of mutations in the cells. The new results were published July 3 in 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."

When scientists want to change the sequence of a stretch of DNA inside cells-either for research purposes or to fix a genetic mutation for therapeutic purposes-they have their choice of two methods. They can use an engineered virus to deliver the new gene to a cell; the cell then integrates the new DNA sequence in place of the old one.

Or scientists can use what's known as custom targeted nucleases, such as TALEN proteins, which cut DNA at any desired location. Researchers can use the proteins to cut a gene they want to replace, then add a new gene to the mix. The cell's natural repair mechanisms will paste the new gene in place.

Previously, Belmonte's lab had pioneered the use of modified viruses, called helper-dependent adenoviral vectors (HDAdVs) to correct the gene mutation that causes sickle cell disease, one of the most severe blood diseases in the world.

He and his collaborators used HDAdVs to replace the mutated gene in a line of stem cells with a mutant-free version, creating stem cells that could theoretically be infused into patients' bone marrow so that their bodies create healthy blood cells.

Before such technologies are applied to humans, though, researchers like Belmonte wanted to know whether there were risks of editing the genes in stem cells. Even though both common gene-editing techniques have been shown to be accurate at altering the right stretch of DNA, scientists worried that the process could make the cells more unstable and prone to mutations in unrelated genes-such as those that could cause cancer.

"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 find out whether this was the case, Belmonte's group, in collaboration with BGI and the Institute of Biophysics, Chinese Academy of Sciences in China, turned to a line of stem cells containing the mutated gene that causes sickle cell disease.

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No extra mutations in modified stem cells

PUBLIC RELEASE DATE:

10-Jul-2014

Contact: Lucky Tran lt2549@cumc.columbia.edu 212-305-3689 Columbia University Medical Center

NEW YORK, NY (July 10, 2014) Columbia University Medical Center (CUMC) researchers have created a way to develop personalized gene therapies for patients with retinitis pigmentosa (RP), a leading cause of vision loss. The approach, the first of its kind, takes advantage of induced pluripotent stem (iPS) cell technology to transform skin cells into retinal cells, which are then used as a patient-specific model for disease study and preclinical testing.

Using this approach, researchers led by Stephen H. Tsang, MD, PhD, showed that a form of RP caused by mutations to the gene MFRP (membrane frizzled-related protein) disrupts the protein that gives retinal cells their structural integrity. They also showed that the effects of these mutations can be reversed with gene therapy. The approach could potentially be used to create personalized therapies for other forms of RP, as well as other genetic diseases. The paper was published recently in the online edition of Molecular Therapy, the official journal of the American Society for Gene & Cell Therapy.

"The use of patient-specific cell lines for testing the efficacy of gene therapy to precisely correct a patient's genetic deficiency provides yet another tool for advancing the field of personalized medicine," said Dr. Tsang, the Laszlo Z. Bito Associate Professor of Ophthalmology and associate professor of pathology and cell biology.

While RP can begin during infancy, the first symptoms typically emerge in early adulthood, starting with night blindness. As the disease progresses, affected individuals lose peripheral vision. In later stages, RP destroys photoreceptors in the macula, which is responsible for fine central vision. RP is estimated to affect at least 75,000 people in the United States and 1.5 million worldwide.

More than 60 different genes have been linked to RP, making it difficult to develop models to study the disease. Animal models, though useful, have significant limitations because of interspecies differences. Researchers also use human retinal cells from eye banks to study RP. As these cells reflect the end stage of the disease process, however, they reveal little about how the disease develops. There are no human tissue culture models of RP, as it would dangerous to harvest retinal cells from patients. Finally, human embryonic stem cells could be useful in RP research, but they are fraught with ethical, legal, and technical issues.

The use of iPS technology offers a way around these limitations and concerns. Researchers can induce the patient's own skin cells to revert to a more basic, embryonic stem celllike state. Such cells are "pluripotent," meaning that they can be transformed into specialized cells of various types.

In the current study, the CUMC team used iPS technology to transform skin cells taken from two RP patientseach with a different MFRP mutationinto retinal cells, creating patient-specific models for studying the disease and testing potential therapies.

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Patient-specific stem cells and personalized gene therapy

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Newswise LA JOLLA-The ability to switch out one gene for another in a line of living stem cells has only crossed from science fiction to reality within this decade. As with any new technology, it brings with it both promise--the hope of fixing disease-causing genes in humans, for example--as well as questions and safety concerns. Now, Salk scientists have put one of those concerns to rest: using gene-editing techniques on stem cells doesn't increase the overall occurrence of mutations in the cells. The new results were published July 3 in 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."

When scientists want to change the sequence of a stretch of DNA inside cells--either for research purposes or to fix a genetic mutation for therapeutic purposes--they have their choice of two methods. They can use an engineered virus to deliver the new gene to a cell; the cell then integrates the new DNA sequence in place of the old one. Or scientists can use what's known as custom targeted nucleases, such as TALEN proteins, which cut DNA at any desired location. Researchers can use the proteins to cut a gene they want to replace, then add a new gene to the mix. The cell's natural repair mechanisms will paste the new gene in place.

Previously, Belmonte's lab had pioneered the use of modified viruses, called helper-dependent adenoviral vectors (HDAdVs) to correct the gene mutation that causes sickle cell disease, one of the most severe blood diseases in the world. He and his collaborators used HDAdVs to replace the mutated gene in a line of stem cells with a mutant-free version, creating stem cells that could theoretically be infused into patients' bone marrow so that their bodies create healthy blood cells.

Before such technologies are applied to humans, though, researchers like Belmonte wanted to know whether there were risks of editing the genes in stem cells. Even though both common gene-editing techniques have been shown to be accurate at altering the right stretch of DNA, scientists worried that the process could make the cells more unstable and prone to mutations in unrelated genes--such as those that could cause cancer.

"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 find out whether this was the case, Belmonte's group, in collaboration with BGI and the Institute of Biophysics, Chinese Academy of Sciences in China, turned to a line of stem cells containing the mutated gene that causes sickle cell disease. They edited the genes of some cells using one of two HDAdV designs, edited others using one of two TALEN proteins, and kept the rest of the cells in culture without editing them. Then, 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.

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No Extra Mutations in Modified Stem Cells, Study Finds