Sickle cell disease (SCD) and β thalassemia are common disorders of the β globin gene (HBB) that arose from the selective pressure of malaria. The β globin gene cluster on chromosome 11 consists of five genes arranged in order of their expression during development: HBE1, embryonic globin; HGB2 and HGB1, fetal γ globin; HBD; and HBB, adult globin. Their sequential expression is controlled by an upstream long-range cis-regulatory element, the locus control region (LCR), as well as other gene-proximal cis elements that recruit transcriptional activator or repressor proteins. Hereditary persistence of fetal hemoglobin (HPFH) is a benign genetic condition caused by mutations that attenuate the switch from fetal γ globin to adult β globin, which results in increased expression of fetal hemoglobin (HbF). Clinical symptoms of SCD or β thalassemia are alleviated in patients who have co-inherited HPFH. The formation of pathologic rigid HbS polymers under deoxygenated conditions in SCD is inhibited by the presence of HbF. These findings prompted a gene manipulation strategy to recapitulate one of the HPFH mutations, which would induce therapeutic levels of HbF.
Dr. Elizabeth Traxler and colleagues focused on a 13-nucleotide deletion from –102 to –114 within the HBG1 promoter, which leads to HbF levels of approximately 30 percent in subjects heterozygous for the mutation. The 13-nucleotide sequence recruits transcriptional repressor proteins, and individuals with wild-type alleles exhibit levels lower than 1 percent HbF. The researchers used CRISPR-Cas9 technology to edit this region in a HUDEP-2 human erythroblast cell line expressing predominantly HbA. They engineered a lentiviral vector to express an mCherry marker, Cas9, and two different nonoverlapping guide RNAs (gRNA-1 and gRNA-2) flanking the target sequence. Transduced cells exhibited an increase in HbF protein; in these cells, there was a commensurate increase in expression of γ globin mRNA and a decrease in expression of β globin mRNA. Mock-infected cultures and control cultures expressing only Cas9 had no HbF. gRNA-1 induced a greater amount of HbF than gRNA-2, but since both were effective, it suggested an on-target rather than an off-target phenomenon.
The next step involved editing peripheral blood CD34+ hematopoietic stem and progenitor cells (HSPCs) from two healthy adults using the same vectors. Transduced cells expressing mCherry were enriched by cell sorting and HbF levels evaluated after erythroid differentiation. Erythroid progeny cells expressing gRNA-1 again produced a better response and showed an increase in HbF levels from 5 percent to 20 percent, and the number of HbF positive cells rose from 18 percent to 58 percent. Relative levels of mRNA confirmed that the switch from γ globin to β globin had been reversed.
These promising results led the research team to test the potential therapeutic effect by transducing CD34+ HSPCs from three patients with SCD using the gRNA-1 lentiviral vector, which increased HbF cells to 90 percent. Culturing these edited cells under hypoxic conditions of 2 percent O2 reduced sickling to 4 percent. Residual HbS polymerization could result from inefficient editing or from mutations that did not induce HbF. To characterize the mutations induced by CRISPR-Cas9 double stranded DNA breaks and error prone nonhomologous end joining, the researchers used polymerase chain reaction and deep sequencing of a 431-nucleotide region encompassing the predicted cleavage sites in the HGB1 or HBG2 genes. This revealed a predominance of the targeted 13-nucleotide deletion and equal mutation rates in the two homologous γ globin genes, as well as 30 other on-target smaller indels. Simultaneous double-stranded DNA cleavage at gRNA-1 recognition sites in the promoters of HBG1 and HBG2 may result in joining of the two ends with loss of the intervening 5.2 kb of genomic DNA. Human CD34+ cells that had been transduced with 10 μg of the Cas9-gRNA-1 vector were examined by quantitative polymerase chain reaction, but there was no evidence of this large detrimental deletion. Bioinformatic analyses predicted several off-target sites, but deep sequencing of these transduced cells showed no indels in the top 15 sites.
In Brief
This study has revealed several important findings: 1) the 13-nucleotide region in the promoters of the HGB1 and HGB2 globin genes is a cis-regulatory element that contains a CCAAT box and a direct repeat, which likely recruits transcriptional repressor proteins; 2) deletion of this region by CRISPR-Cas9 genome editing partially reversed the switch from fetal to adult globin in CD34+ HSPCs from normal individuals and patients with SCD; and 3) increased expression of HbF in edited SCD cells inhibited polymerization of HbS and the pathologic sickling of these cells under hypoxic conditions.
These exciting results thus provide proof of principle for a therapeutic genome editing strategy whereby a benign naturally occurring mutation is recapitulated ex vivo in cells from patients with SCD. This study provides a framework for further refinement of the editing procedure and the application to other β hemoglobinopathies.
Competing Interests
Dr. Coetzer indicated no relevant conflicts of interest.
