NEW YORK (GenomeWeb) – A new study led by scientists from St. Jude Children's Research Hospital provides yet another strategy for alleviating hereditary hemoglobin deficiencies like beta-thalassemia and sickle cell disease using CRISPR/Cas9 genome editing.
Along with scientists from the University of Pennsylvania, the Children's Hospital of Philadelphia, Pennsylvania State University, Oxford University, and Japan's University of Tsukuba, first author Elizabeth Traxler and senior author Mitchell Weiss, both of St. Jude's, described how they mutated a 13 base pair stretch of promoters for subunits of fetal hemoglobin — hemoglobin subunit gamma 1 and 2 (HBG1 and HBG2).
Specifically, the scientists were able to edit patient-derived hematopoietic stem and progenitor cells (HSPCs). The scientists said their edited cells produced levels of fetal hemoglobin that led to reduced sickle cell morphology when differentiated into red blood cells. They published their study today in Nature Medicine.
Usually, expression of HBG1/2 decreases as adult hemoglobin expression increases; however, some people have benign mutations that cause blood cells to continue fetal hemoglobin production over their entire life. "Individuals with genetic mutations that persistently elevate fetal hemoglobin are resistant to the symptoms of sickle cell disease and beta-thalassemia, genetic forms of severe anemia that are common in many regions of the world," Weiss said. "We have found a way to use CRISPR gene editing to produce similar benefits."
They join several other teams looking to spur fetal hemoglobin production using gene editing, especially CRISPR/Cas9. In September 2015, scientists from Dana Farber Cancer Institute and the Massachusetts Institute of Technology, led by Stuart Orkin and Feng Zhang, published a study in Nature describing CRISPR/Cas9-based editing of a promoter region of the enhancer gene BCL11A, an important gene in the fetal-to-adult hemoglobin transition.
Perhaps the most promising aspect of the new study is that it mimics mutations already seen in humans. Several different mutations in regulatory regions can lead to expression of fetal hemoglobin. For example, a single base pair substitution can form a binding site for a transcriptional activator or eliminate cis elements that recruit proteins to repress HBG1/2, the authors wrote.
The scientists focused on a 13-base pair deletion occasionally found in the HBG1 promoter region, which eliminates both a CCAAT box and a direct repeat that recruit repressor proteins. It was just one of dozens of small indel mutations caused by the non-homologous end-joining repair pathway, but by combing through hundreds of colonies derived from clones of edited hematopoietic stem cells, they identified several instances of the specific mutation.
Analysis with flow cytometry and immunostaining suggested that the cells did indeed produce fetal hemoglobin.
While HBG2 has an identical promoter region as HBG1, the exact 13 base pair deletion is not naturally found in that region, though other mutations in that region are associated with long-term production of fetal hemoglobin. This leaves open the possibility that the guide RNA used to target the HBG1 promoter would induce edits at both locations, leading to a large deletion of over 5 kilobases.
"Our work has identified a potential DNA target for genome editing-mediated therapy and offers proof of principle for a possible approach to treat sickle cell and beta-thalassemia," Weiss said. However, the scientists said they still needed to fully characterize off-target editing and perform head-to-head comparisons with other gene-editing strategies to treat these diseases.