The promise of a revolutionary gene-editing technology is beginning to be realized in experiments aimed at curing sickle cell disease.
Scientists reported Wednesday that they used the CRISPR-Cas9 system to correct a tiny genetic mutation that causes the blood disease, which affects millions of people around the world.
So far, the feat has been demonstrated only in human cells that were confined to laboratory dishes. But in a promising step, the researchers used the same DNA-editing technique to alter human cells that were transfused into mice. After 16 weeks, the mice still had cells that contained the edited gene, according to a study in the journal Science Translational Medicine.
"What we have right now, if we can scale it up and make sure it works well, is already enough to form the basis of a clinical trial to cure sickle cell disease with gene editing," said study leader Mark DeWitt, a postdoctoral fellow at UC Berkeley's Innovative Genomics Initiative.
Sickle cell disease, or SCD, occurs when a single letter in the HBB gene is a T instead of an A. That makes the disease an ideal candidate for an intervention using gene-editing technology, experts say.
“It is one single target that is the same in every sickle cell patient,” said Dana Carroll, a biochemist at the
Healthy red blood cells resemble a disk with a depression in the center. But the error in the HBB gene causes people to produce red blood cells that are shaped like a C, or sickle. These cells are less flexible than regular red blood cells and can restrict the flow of oxygen to the body's tissues, causing severe pain and, over time, organ damage.
Red blood cells are constantly being produced by stem cells in our bone marrow, and the only lasting way to cure sickle cell disease is with a bone marrow transplant. But few people choose to go this route. It requires finding a matched donor, an option that isn't always available. If one is found, the patient must have chemotherapy to clear out his own immune system, then take immunosuppressant drugs to reduce the risk that his body will reject the foreign tissue.
"It's painful and dangerous to do a bone marrow transplant," Carroll said.
That's why a gene-editing solution is so attractive. The hope is that doctors will be able to remove a patient's stem cells, alter their DNA in the lab, then put the cells back in the body, where they will produce healthy red blood cells indefinitely.
CRISPR-Cas9 has transformed gene editing by making it easier and cheaper to alter a cell's DNA. It combines a DNA-cutting protein with a small piece of RNA that guides the protein to just the right spot. Once the DNA strands have been broken, a corrected version of the code can be spliced in.
But the system is still quite new — the first report of CRISPR being used to alter living cells came in 2013.
So far, scientists have discovered that some cells require more work to edit than others.
"It's very easy to get CRISPR material into cells that have been in culture for a long time and are growing rapidly," Carroll said. "But the cells that lead to the continuous production of new red blood cells are harder to modify."
Researchers are not sure why that is, but part of the goal of the new study was to determine the best way to get CRISPR into stem cells so the desired edit could be made.
The team accomplished this using a technique called electroporation, which uses electricity to open the pores of a cell's membrane.
"Basically, we give the cells a very quick electrical shock that helps get the material outside the cell to move inside the cell," Carroll said.
In one of their experiments, the researchers took stem cells from patients with sickle cell disease and used CRISPR-Cas9 to snip out the section of the HBB gene with the faulty mutation, then replaced it with a corrected version. The trials were successful, but they did not generate enough cells to test them in mice.
The researchers also took stem cells from healthy patients and used the same technique to splice the disease-causing mutation into the HBB gene.
"That sounds sort of nuts, but it is hard to get cells from sickle cell patients," Carroll said. "It was easier for us as we were working out the technology to get normal cells and convert those to sickle."
In some of their most successful trials, up to 40% of the cells were successfully edited and another 40% had a scar at the editing site, though no fix was made. The final 20% remained unchanged.
In the world of genetic editing, those are pretty good results, Carroll said.
The researchers wanted to see how altered human cells would survive in the bodies of mice. So they transplanted roughly 1 million of the cells into each mouse in the study. For these experiments, about 12% of the stem cells had been fully altered by CRISPR.
After 16 weeks, the researchers tested human blood cells in the bone marrow of five mice. They found that, on average, 2.3% of these cells had the edited DNA with the SCD trait.
The researchers said that if they had done the conversion in reverse and fixed the SCD mutation, it should have worked just as well.
"The change we are making is identical to the one that we hope to make in humans, except for the direction," Carroll said.
Previous research has shown that even a small change in the percentage of stem cells that produce healthy red blood cells can have a positive effect on a person with sickle cell disease. Still, experts say 2% is not enough.
"This is an important incremental step in bringing this sort of gene therapy to the clinic, but you need a little higher rate of correction to make it a therapy that is likely to be successful," said Dr. John Strouse, a Duke University hematologist and member of the American Society of Hematology Sickle Cell Disease Task Force.
"You don't need to correct 100% of the cells to cure the disease, but you might need 5% or 10%," said Strouse, who wasn't involved in the new study.
DeWitt said that the team's next goal is to increase the percentage of altered cells that are present after 16 weeks from 2% to 5%.
In addition, before clinical trials can begin, the team will have to figure out a way to scale up the ability to edit genes. While each mouse received 1 million cells, a human would need hundreds of millions of cells.
Finally, they'll have to make sure that CRISPR doesn't alter other parts of the genome by accident.
"It will be years of very careful and rigorous study to ensure that it's totally and completely safe before we even contemplate an actual clinical trial," DeWitt said. "But we have a path. We just have to walk down it."
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