'Not just on the chalkboard'

STANFORD, Calif.—CRISPR/Cas9 gene editing has become one of the most intriguing technological advances in genomics for the ability it offers to knock out and edit genes, and Stanford University School of Medicine researchers have showed that there's potential in its promise. In preclinical models of mice, a team was able to correct the mutation that causes sickle cell disease and transplant healthy, corrected hematopoietic stem cells in mice.

The mutation in the HBB gene that typifies sickle cell disease causes red blood cells to produce an altered version of hemoglobin, which is the primary component of red blood cells and what enables them to absorb oxygen from the lungs and transfer it to the tissues of the body. This mutation makes the red cells take on a sickle shape when oxygen levels are low. In addition to the abnormal shape, sickle cells are rigid and sticky, leading them to clog blood vessels, which in turn can result in pain and organ damage. These deformed cells also die faster than normal, healthy red blood cells—within an average of 10 days—which often leads to anemia, another cause of organ damage.

CRISPR, or clustered, regularly interspaced, short palindromic repeat, refers to a pattern of DNA sequences that appear in bacterial DNA and are thought to reflect evolutionary responses to past viral attacks. The Cas9 protein is a nuclease, an enzyme that cuts DNA in two places. Using the two together offers significant specificity in gene editing, allowing scientists to isolate genes to determine their function and the effects of knocking them out, and, in the case of this work, to cut out DNA fragments and replace them with a corrected genetic sequence.

“What we’ve finally shown is that we can do it,” said Dr. Matthew Porteus, associate professor of pediatrics at Stanford and senior author on this recent work. “It’s not just on the chalkboard. We can take stem cells from a patient and correct the mutation and show that those stem cells turn into red blood cells that no longer make sickled hemoglobin.”

Porteus' team started their work with hematopoietic stem cells from the blood of sickle cell disease patients. They corrected the HBB gene mutation using CRISPR and then concentrated the stem cells so that 90 percent of them carried the corrected sickle cell gene.

Specifically, the team's approach consisted of “a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells,” as noted in the paper's abstract. This tactic, the authors note, shows “efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult β-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles.”

The altered cells were then injected into young mice. Sixteen weeks after injecting them, upon examination, the team found the corrected stem cells were flourishing in the mice's bone marrow. Compared to regular sickle cells, the corrected cells had the same lifespan as normal red blood cells, about four months or 120 days, and seemed to behave like normal hematopoietic stem cells.

The encouraging part, as noted by Porteus, is that the corrected cells don't have to replace all of the original sickle cells—so long as the proportion of sickle cells to healthy red blood cells is below 30 percent, patients present with no symptoms of their disease.

Despite the promise of CRISPR, one of the ongoing issues is that no CRISPR-edited genes have been tested for safety or efficacy in human trials as of yet. It's something of a catch-22; because none have been tested, there's no knowing if edited genes could cause immune reactions or off-target effects—and because of that risk, none of these approaches has seen human trials.

Porteus remarked that “The consensus in the field is that there’s no one test we can do to prove that something is safe. We can’t just say, ‘Oh, just run this test, and that’ll show if it’s safe or not.’ That test doesn't exist.” Despite this, he is “excited about working to eventually bring this type of therapy to patients.”

And there are many patients that could benefit from such a therapy. At present, roughly 70,000 to 100,000 people in the United States suffer from sickle cell disease, with millions affected globally. While children in high-income countries generally survive with this disease, those in low-income countries often die before age 5. Current treatment options for patients with sickle cell disease consist of blood and bone marrow stem cell transplantation.

The results of this work were shared in a paper titled “CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells,” which was published online in Nature. Postdoctoral scholars Drs. Daniel Dever and Rasmus Bak are the lead authors.

This research received support from the National Institutes of Health (grants PN2EY018244, R01AI097320 and R01AI120766), the Stanford Child Health Research Institute, the Laurie Kraus Lacob Scholar Award in Pediatric Translational Research and the Laurie Kraus Lacob Endowment Fund, as well as Stanford’s Department of Medicine.

SOURCE: Stanford University School of Medicine press release