Scientists in the United States have used CRISPR-Cas9 technology to edit the genomes of a specific subset of blood forming stem cells as an approach to reversing the clinical symptoms of blood disorders such as sickle cell disease (SCD) and beta-thalassemia. Beta-hemoglobinopathies are caused by gene mutations that result in abnormal production of hemoglobin. Rather than try to repair the faulty gene for adult hemoglobin, the Fred Hutchinson Cancer Research Center-led team’s research in nonhuman primates used CRISPR-Cas9 editing to take the genetic brakes off the cells’ production of fetal hemoglobin. This form of hemoglobin is produced in the fetus and in newborns, but is normally just about completely turned off shortly after birth, when the body switches over to production of adult hemoglobin.

Results from the proof-of-principle study suggest that it may be possible to harness CRISPR-Cas9 technology to introduce relevant gene modifications into only a specific subset of stem cells that then differentiate into fetal hemoglobin-producing blood cells. Modifying this subset of stem cells would effectively reduce the costs of gene-editing treatments for blood disorders, while minimizing the risks of off-target effects.

The study represents the first time that scientists have specifically edited the genetic makeup of a specialized subset of adult blood stem cells that are the source of all cells in the blood and immune system.”Targeting this portion of stem cells could potentially help millions of people with blood diseases,” said Hans-Peter Kiem, PhD, director of the stem cell and gene therapy program and a member of the Clinical Research Division at Fred Hutchinson Cancer Research Center. Kiem’s team collaborated with researchers across the U.S., and in Ireland. “Not only were we able to edit the cells efficiently, we also showed that they engraft efficiently at high levels, and this gives us great hope that we can translate this into an effective therapy for people.”

Kiem holds the Stephanus Family endowed chair for cell and gene therapy, and is senior author of the team’s published paper in Science Translational Medicine, titled “Therapeutically relevant engraftment of a CRISPR-Cas9–edited HSC-enriched population with HbF reactivation in nonhuman primates.”

Beta-hemoglobinopathies such as SCD and beta-thalassemia are among the most common genetic disorders caused by mutations in a single gene. The only curative treatment is transplantation of donor blood stem cells, but there aren’t enough donors, and treatment isn’t without potential complications. The clinical symptoms of beta-hemoglobinopathies are naturally less severe, however, in patients who have genetic mutations that cause them to continue to produce fetal hemoglobin, a form of hemoglobin that is normally only produced for a few months after birth, after which the body switches over to full production of adult hemoglobin. This benign genetic condition is known as hereditary persistence of HbF (HPFH). The ability of fetal hemoglobin to act as a substitute for adult hemoglobin, points to alternative strategies for treating disorders such as SCD and beta-thalassemia. “Reactivation of fetal hemoglobin (HbF) is being pursued as a treatment strategy for hemoglobinopathies,” the authors pointed out.

For their reported studies the researchers isolated populations of nonhuman primate CD34 receptor-expressing hematopoietic stem and progenitor cells (HSPCs), and a more targeted population of CD34+CD90+CD45RA hematopoietic stem cells (HSCs). They then used CRISPR-Cas9 technology to introduce mutations that recapitulate mutations typically found in HPFH, and transplanted either edited CD34+ HSPCs, or the more targeted CD90+CD45RAcells “comprising less than 10% of the total CD34+ cell number” into rhesus macaque monkeys. Initial tests confirmed that up to 78% of the cells took up the edits, and after transplantation into the animals the cells engrafted successfully. Editing just the CD34+CD90+CD45RA cells effectively meant that the researchers could reduce the target cell count by more than 10 fold. “… solely editing only the CD90+CD45RA- population achieved comparable in vivo editing relative to the current clinical gold standard targeting the entire CD34+ cell population.”

An analysis of the macaques’ bone marrow (BM) six months after they received the edited HSPC or targeted HSC cell transplants confirmed that the engrafted cells differentiated into the complete range of blood cells, with evidence that the CRISPR-generated gene mutation persisted. “… the gene-edited CD34+CD90+CD45RA population as well as HSC-enriched cells are capable of homing and repopulating the BM stem cell compartment to generate a balanced output of gene-edited lymphoid, myeloid, and erythroid blood cells,” the team wrote.

About 30% of the blood cells contained the edits, resulting in nearly 20% of red blood cells derived from the targeted HSC population containing fetal hemoglobin. The authors suggest that the successful persistence of editing of this much smaller population of HSC cells means the approach could be more cost effective at scale up. “Edited cells effectively and stably reactivated HbF, as evidenced by up to 18% HbF-expressing erythrocytes in peripheral blood,” the authors wrote. “Similar results were obtained by editing highly enriched stem cells, defined by the markers CD34+CD90+CD45RA, allowing for a 10-fold reduction in the number of transplanted target cells … thus circumventing issues associated within scale-up and considerably reducing the need for editing reagents without affecting hematopoietic recovery, engraftment, or HbF reactivation.”

The rhesus animals were monitored for up to a year and a half, with no evidence of adverse effects on the composition of the mature blood cell lines. There was also no evidence of off-target effects. “Since the CRISPR technology is still in early stages of development, it was important to demonstrate that our approach is safe,” commented Olivier Humbert, PhD, first author of the published paper and a staff scientist at the Kiem lab. “We found no harmful off-target mutations in edited cells and we are currently conducting long-term follow-up studies to verify the absence of any undesired effect.”

The authors maintain that their genome editing approach has the potential to offer “a curative option for patients with hemoglobinopathies by introducing targeted, naturally occurring HPFH mutations … These results represent a substantial improvement in engraftment of gene-edited CD34+ HSPCs as compared to our previous investigations using bulk cells treated with zinc finger nucleases or TALENs in the NHP model.” They conclude that “the extent of in vivo gene editing achieved in our study using bulk CD34+ or the CD34+CD90+CD45RA subpopulation should be within a therapeutically relevant range for a number of genetic diseases.”

Kiem is hopeful that their results could pave the way to a future human therapy for relevant hemoglobinopathies. “Twenty percent of red blood cells with fetal hemoglobin— what we saw with this method—would be close to a level sufficient to reverse symptoms of sickle cell disease.” The authors suggest that the gene editing technique could also feasibly be used in other diseases. “By demonstrating how this select group of cells can be efficiently edited for one type of disease, we hope to use the same approach for conditions such as HIV and some cancers,” Kiem suggested.

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