Base editing—the genome editing technique that alters base pairs using a nuclease-deficient Cas9 fused to a deaminase—was found to restart fetal hemoglobin expression in sickle cell disease (SCD) patient cells. This result stems from work that compared five genome editing strategies, in CD34+ hematopoietic stem and progenitor cells, using either Cas9 nuclease or adenine base editors.

The findings suggest that adenosine base editing raised the expression of fetal hemoglobin to higher, more stable, and more uniform levels than other genome editing technologies that use CRISPR/Cas9 nuclease in human hematopoietic stem cells.

This study was published in Nature Genetics in the paper, “Potent and uniform fetal hemoglobin induction via base editing.

“Ultimately, we showed that not all genetic approaches are equal,” said Jonathan Yen, PhD, genome engineering group director at St. Jude Children’s Research Hospital. “Base editors may be able to create more potent and precise edits than other technologies. But we must do more safety testing and optimization.”

SCD and beta-thalassemia are blood disorders caused by mutations in the gene encoding hemoglobin affecting millions of people. Restoring gene expression of an alternative hemoglobin subunit active in a developing fetus has previously shown therapeutic benefit in SCD and beta-thalassemia patients. The researchers wanted to find and optimize genomic technology to edit the fetal hemoglobin gene.

Adult hemoglobin, expressed primarily after birth, contains four protein subunits—two beta-globin and two alpha-globin. Mutations in the beta-globin gene cause sickle cell disease and beta-thalassemia. But humans have another hemoglobin subunit gene (gamma-globin), which is expressed during fetal development instead of beta-globin. Gamma-globin combines with alpha-globin to form fetal hemoglobin. Normally around birth, gamma-globin expression is turned off, and beta-globin is turned on, switching from fetal to adult hemoglobin. Genome editing technologies can introduce mutations that turn the gamma-globin gene back on, thereby increasing fetal hemoglobin production, which can effectively substitute for defective adult hemoglobin production.

“The gamma-globin [fetal hemoglobin] gene is a good target for base editing because there are very precise mutations that can reactivate its expression to induce expression after birth, which may provide a powerful ‘one-size-fits-all’ treatment for all mutations that cause SCD and beta-thalassemia,” said Mitchell Weiss, MD, PhD, chair of the department of hematology at St. Jude.

One alteration installed by adenosine base editing was particularly potent for restoring fetal hemoglobin expression in post-natal red blood cells, increasing fetal hemoglobin to meaningful levels. The authors describe the most potent modification as the generation of γ-globin –175A>G. Homozygous –175A>G edited erythroid colonies, they wrote, “expressed 81 ± 7% HbF versus 17 ± 11% in unedited controls, whereas HbF levels were lower and more variable for two Cas9 strategies targeting a BCL11A binding motif in the γ-globin promoter or a BCL11A erythroid enhancer.”

“We looked closely at the individual DNA sequence outcomes of nucleases and base editors used to make therapeutic edits of fetal hemoglobin genes. Since nucleases often generate complex, uncontrolled mixtures of many different DNA sequence outcomes, we characterized how each nuclease-edited sequence affects fetal hemoglobin expression. Then we did the same for base editing outcomes, which were much more homogeneous,” said David Liu, PhD, professor at the Broad Institute who discovered base editing.

The study discovered that using base editing at the most potent site in the gamma-globin promoter achieved 2- to 4-fold greater HbF levels than Cas9 editing. They further demonstrated that these base edits could be retained in engrafting blood stem cells from healthy donors and SCD patients by putting them into immunocompromised mice.

“In our comparison, we found unanticipated problems with conventional Cas9 nucleases,” Weiss said. “We were somewhat surprised that not every Cas9 insertion or deletion raised fetal hemoglobin to the same extent, indicating the potential for heterogeneous biological outcomes with that technology.”

Individual red blood cells derived from hematopoietic stem cells treated with the same Cas9 produce a more variable amount of fetal hemoglobin compared to cells treated with base editing. Thus, base editing produced more potent, reliable, and consistent outcomes, which are desirable therapeutic properties.

Though base editing performed well, researchers have yet to determine its safety in patients. Notably, base editing may have some risks not presented by Cas9; for example, some early base editors can cause undesired changes in genomic DNA or RNA at off-target sites. However, when compared for safety, base editing caused fewer genotoxic events, such as p53 activation and large deletions. Base editing was much more consistent in its edits and products—a highly desirable safety property for a clinical therapy. In contrast to conventional Cas9, which generates uncontrolled indels, base editing generates precise nucleotide changes with few undesired byproducts.

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