Knocking out BCL11A switches cellular production of hemoglobin from mutated adult form to normal fetal form.
Scientists have managed to correct sickle cell disease (SCD) in adult mice by knocking out a single gene to enable production of the fetal form of hemoglobin (HbF). The investigators, led by a team at Boston Children’s Hospital, the Dana-Farber Cancer Institute, and the Howard Hughes Medical Institute, found that silencing BCL11A (a gene that codes for one of the transcriptional repressors of HbF expression) turned HbF production back on in adult mice, and the animals generated enough of this form of hemoglobin to correct the hematologic and pathologic defects associated with SCD.
Stuart H. Orkin, M.D., at the Dana-Farber/Children’s Hospital Cancer Center, and colleagues say their results demonstrate that BCL11A might serve as a target for treating SCD and related blood disorders such as thalassemias, even though it’s almost certainly not the only factor involved in regulating HbF production. “I think we’ve demonstrated that a single protein in the cells is a target that, if interfered with, would provide enough fetal hemoglobin to make patients better,” Dr. Orkin states. “It’s been hypothesized for three decades that fetal hemoglobin could be turned on once we understood the mechanism of hemoglobin switching, and this is the first evidence of a target to do that.”
The team’s results are published in Science in a paper titled “Correction of Sickle Cell Disease in Adult Mice by Interference with Fetal Hemoglobin Silencing.”
The fetal form of hemoglobin (comprising two α and two γ subunits) is produced by the growing fetus from about two months into gestation, and its production continues after birth but is gradually replaced by the adult form of hemoglobin (HbA, which carries two α subunits and two β-globin subunits, instead of the two γ-globins), the researchers explain. Once the infant hits 3–6 months of age, fetal hemoglobin has generally dwindled almost altogether, although the level of HbF that continues to be produced by adult individuals is inherited as a quantitative trait.
SCD, meanwhile, is caused by a single amino acid substitution in one of the beta-globin subunits of adult hemoglobin, and the resulting mutant hemoglobin (HbS) polymerizes, interferes with oxygen carriage, and causes red blood cells to become sickle-shaped and accumulate in small blood vessels. Because the sickle cell mutation only affects adult hemoglobin, the symptoms of sickle cell disease don’t become manifest until the infant has switched over from production of HbF. Interestingly, SCD patients who naturally produce the most HbF generally suffer less severe symptoms of their disease.
The involvement of BCL11A in the regulation of HbF production was initially highlighted by genome-wide association studies (GWAS), and work by Dr. Orkin’s team has also shown that knocking out BCL11A in cultured human red cell progenitors activates fetal hemoglobin and also silences production of the mutated adult hemoglobin.
To see whether these findings could be translated into benefits in vivo, the researchers first knocked out the BCL11A gene specifically in the erythroid cells of mice expressing the human beta-globin gene cluster (β-YAC mice) to see what the effects were on γ-silencing. Importantly, the conditional knockout animals demonstrated normal erythropoiesis in fetal liver and adult bone marrow, despite the absence of BCL11A. The researchers also found that, as with conventional knockouts, hemoglobin switching didn’t occur in fetal liver, but started to decline after birth, such that levels of γ-globin declined over time to a residual level of ~11% in adult mice of 30 weeks and older.
Transcription profiling of erythroid cells from the adult bone marrow of both BCL11A-null and wild-type mice showed no difference in the expression of known erythroid transcriptional regulators, including GATA1, FOG1, NF-E2, KLF1, SOX6, and MYB. However, mouse embryonic β-like and α-like globin genes were differentially expressed. “Thus, BCL11A is highly selective in controlling targets in erythroid cells, and only expression of the globin genes is substantially affected in its absence,” the researchers write.
The studies had yet to demonstrate whether γ-globin genes that are fully silenced during normal development can be reactivated upon loss of BCL11A. The researchers therefore generated β-YAC mice in which the BCL11A gene was excised on administration of interferon. In these animals, inactivation of BCL11A led to re-expression of the developmentally silenced γ-globins to 13.8% of total β-like human globins within just a week, and these levels were subsequently maintained.
However, the partial silencing of γ-globin expression in BCL11A-null erythroid cells does indicate that additional silencing mechanisms are at work independently of BCL11A, the team notes. Two potential epigenetic mechanisms are DNA methylation and histone deacetylation, which have both previously been implicated in HbF control.
Analysis did indeed show that DNA methylation of the γ-globin promoters progressively increased in BCL11A-null erythroid cells. While administration of the DNA methylation inhibitor 5-azaD to normal β-YAC mice led to a very small increase in γ-globin mRNA, treating the BCL11A knockout mice with 5-azaD boosted γ-globin mRNA expression up to 37.9%. Conversely, administration of the HDAC inhibitor SAHA seemed to work alongside BCL11A expression to depress γ-globin mRNA. “These results show that loss of BCL11A markedly enhances the effects of DNA demethylation and HDAC inhibitors in reactivating HbF expression, and suggest that BCL11A down-regulation might be combined with known HbF inducers for efficient HbF augmentation,” the investigators state.
The work to this point supported the notion that BCL11A might represent a target for reactivating HbF in sickle cell disease, but hadn’t evaluated the effects of BCL11A silencing on disease in vivo. To this end they turned to Berkeley SCD mice, which express only human fetal, and adult sickle cell hemoglobin, and display similar SCD symptoms to human patients, including severe hemolytic anemia, reticulocytosis, and shortened RBC survival.
Introducing BCL11A-null alleles into these animals resulted in the correction of hematologic parameters including RBC counts and hemoglobin content. Importantly, and in contrast to SCD mice that retained BCL11A, the SCD/Bcl11a-/- knockouts didn’t develop sickled cells, and demonstrated much improved red cell survival. Spleen size, which is a measure of compensatory erythropoiesis in SCD, was also dramatically reduced in the knockout animals, and urine osmolality was normal, indicating improved renal function (in SCD, RBC sickling leads to reduced medullary blood flow and impairs urine-concentrating ability). Organ histopathology characteristic of SCD was in addition reversed in SCD/Bcl11a–/– mice.
Critically, expression of γ-globin genes was greatly elevated in adult SCD/Bcl11a–/– mice, and reached 28.3% of total β-like human globins, compared with 1.3% in SCD mice. And while staining assays showed that the peripheral blood of control and SCD mice contained between about 3% and 7% of HbF containing cells, the peripheral blood of SCD/Bcl11a–/– mice exhibited strong pancellular staining of HbF, and F-cells accounted for 85.1% of total red blood cells. Similar results were obtained when an independent strain of SCD mouse was used.
The level of HbF expression in the absence of BCL11A thus “exceeds the estimated ~15–20% HbF thought to be necessary to virtually eliminate SCD phenotypes in patients,” the researchers write. “We anticipate that our findings should apply similarly to the β-thalassemias.”
Of particular significance was the observation that switching on the production of HbF correlated with a parallel reduction in levels of the mutant adult form of hemoglobin. “The red blood cell lineage performs a balancing act when it comes to manufacturing hemoglobin,” Dr. Orkin explains. “Increasing the production of one form reduced production of the other.”
The resarchers admit that there will be “formidable barriers” to overcome before their results can be applied in human therapeutics, not least because as a transcription factor, BCL11A represents a “challenging” target. Nevertheless, they state, interfering with BCL11A expression and/or function could feasibly be achieved using strategies such as RNA inhibitory molecules or somatic gene transfer of shRNA.
In the meantime, Dr. Orkin’s team is working to provide deeper insights into the workings of BCL11A, and the translation of their studies in mice. “We want to better understand the network of genes and proteins that interact with BCL11A to see if any of them might also stand out as targets,” Dr. Orkin remarks. “We are also screening the protein against libraries of chemicals in the hope of identifying those that interfere with it.”