ALBERT

Description: During human ontogeny, high-level transcription within the beta-globin gene cluster switches sequentially from embryonic-to-fetal-to-adult genes. Beta-thalassemias and sickle-cell disease are manifested by reduced or mutated expression of the adult-stage, beta-globin gene. Research is aimed toward the eventual therapeutic goal of safely preventing or reversing the fetal-to-adult hemoglobin switch among these patient populations. To identify genes that may be involved in regulation of the fetal-to-adult erythroid switch, purified CD34(+) cells from six umbilical cord (fetal) and six adult peripheral blood samples were cultured in serum-free medium, and gene expression libraries were prepared and sequenced from CD71(+), CD235a(+) erythroblast mRNA. In total, 546 million paired-end reads with a length of 101bp were generated for a comparison of cord and adult erythroblast transcriptomes. Reads were aligned to the human reference genome (hg19), and differential gene expression was identified [false discovery rate ≤ 0.05, fold change ≥ 1.5, and reads per kilobase per million mapped reads (RPKM) ≥ 0.5]. A total of 145 genes were differentially expressed according to these criteria, with four of the top five encoding targets of the let-7 family of microRNAs. The topmost gene was insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), which is normally involved in transcriptome regulation and developmental timing. IGF2BP1 expression was 770-fold increased in the fetal erythroblasts (RPKM 〉 3.0) compared with low background levels in adult erythroblasts (RPKM 〈 0.01). IGF2BP1 protein is present in fetal tissues including fetal liver; however, it is not detected in adult human bone marrow. A potential role for adult-stage IGF2BP1 over-expression (IGF2BP1-OE) in the regulation of globin genes and proteins was explored using lentiviral vectors designed for let-7 resistant, erythroid-specific expression of IGF2BP1 protein. IGF2BP1-OE transduced CD34(+) cells expressed the transgenic protein and maintained their ability to differentiate, accumulate hemoglobin, and enucleate ex vivo in the presence of erythropoietin. Globin mRNA and protein levels were investigated. While alpha-globin mRNA remained unchanged, gamma-globin mRNA became predominant [90% of (gamma + beta) mRNA] in IGF2BP1-OE samples [Control (empty vector) = 3.2E+06 ± 8.2E+05 copies/ng; IGF2BP1-OE = 2.0E+07 ± 5.9E+06 copies/ng; p 〈 0.05], and beta-globin mRNA decreased to minor levels [Control (empty vector) = 2.2E+07 ± 4.0E+06 copies/ng; IGF2BP1-OE = 2.2E+06 ± 6.2E+05 copies/ng; p 〈 0.05]. IGF2BP1-OE caused a pan-cellular HbF distribution by flow cytometry. Cellular fetal hemoglobin percentages [HbF/(HbF + HbA)] were measured as 5.3 ± 0.4% in donor matched control cells versus 80.3 ± 3.7% in IGF2BP1-OE cells (p 〈 0.05). HPLC tracings revealed that the minor HbA2 peak, composed of alpha and delta globin chains, was reduced or absent in IGF2BP1-OE. Also, IGF2BP1-OE suppressed the expression of related genes including the transcription factor BCL11A. These data demonstrate that erythroblast IGF2BP1 is silenced in humans during fetal-to-adult ontogeny, and that IGF2BP1 in adult erythroblasts reverses the developmentally related switch in beta-like globin gene and protein expression patterns. Disclosures No relevant conflicts of interest to declare.

Description: Reticulocytosis begins early in life in infants with sickle cell anemia (HbSS, SCA) as fetal hemoglobin (HbF) is replaced by sickle hemoglobin (HbS). Almost one quarter of children with SCA will have at least one episode of splenic sequestration before the age of 2 years, and immature (CD36+) reticulocytes released into circulating blood may contribute to this early pathology because of splenic trapping of those cells. Additionally, clinical studies have identified that absolute reticulocyte counts (ARC) greater than 200 K/µL during early infancy (60-196 days of age) may be a useful marker to identify SCA infants who are at the highest risk for SCA-associated events during childhood. The objective of this study was to determine if expression of the adhesion marker named CD36 changes with ARC levels during early childhood in pediatric SCA patients. Infants between the ages of 6 and 12 months were enrolled in a prospective, longitudinal, observational study after written consent was obtained from a parent or legal guardian. Participants were not receiving hydroxyurea or monthly blood transfusions. After consent was provided, peripheral blood was obtained during study visits at steady state and analyzed within 48 hours of collection and storage at 40 C. Steady state was defined as a sample drawn at least 30 days following an acute event and at least 60 days following a blood transfusion. Hematologic data, including ARC and HbF levels, were measured using CLIA approved methods. F-cells were enumerated with flow cytometry following intracellular staining with a HbF fluorescent antibody. Reticulocytes were identified by flow cytometry after thiazole orange staining and were further quantitated with CD36, CD45, CD71 and CD235a staining. CD36+ reticulocytes were defined here by surface CD36 detected at levels greater than two standard deviations above unstained controls. Calculations were made to determine the proportion of total circulating reticulocytes that expressed surface CD36+ as well as the absolute number of circulating CD36+ reticulocytes/microliter of whole blood. Correlations were calculated to determine the relationships of ARC with HbF, F-cells, and other hematologic data, while two-tailed t-tests were used to compare means. Mean age at enrollment was 278±70 days and 62.5% of the participants were male. None of the eight enrolled patients had SCA complications or hospitalizations prior to enrollment. Mean HbF and F-cell levels were 40.8±11.1% and 92.8±8.8%, respectively. Mean hemoglobin was 10±0.6 g/dL and ARC 189.1±69.4 K/µL and mean number of absolute CD36+ red blood cells was 29.3±19.1 K/µL. ARC increased over time in 6 of the 8 (75%) participants for the study observation period. ARC was positively correlated with the proportion of CD36+ reticulocytes (r=0.78, p

Description: Pharmacologic and genetic regulation of fetal hemoglobin (HbF) remains a major goal for treatment of the beta-thalassemias and sickle cell disease. Recently, the let-7 microRNA heterochronic pathway of RNA-binding factors was shown to be developmentally correlated with HbF expression in humans. Experimental manipulation of the pathway components (LIN28, let-7, and the IGF2 binding proteins) demonstrated moderate increases in fetal hemoglobin (around 15-35% HbF) with the exception of IGF2BP1, which caused a nearly complete reversal in gamma- and beta-globin gene expression resulting in HbF levels of 60-70% among cultured adult human erythrocytes. To further explore IGF2BP1 effects on HbF and erythropoiesis, lentiviral transduction of CD34(+) cells was performed with let-7 resistant expression of IGF2BP1 driven by the human SPTA1 gene (IGF2BP1-OE). Donor-matched control transductions were studied for comparison. For protein localization studies, confocal imaging of sorted cells was utilized. IGF2BP1-OE caused IGF2BP1 protein expression throughout the cytoplasm and localized to small granules. Granules were unevenly distributed and more abundantly observed in the perinuclear regions. Nuclear detection of IGF2BP1 protein remained at background control levels. We thus hypothesized that IGF2BP1 protein in these cytoplasmic granules may regulate globin gene expression by binding RNAs that encode epigenetic modifiers of the beta-globin locus. To test this hypothesis, RNA-immunoprecipitation (RIP) was performed using a RIP-certified anti-IGF2BP1 antibody. Next generation sequencing and qRT-PCR were used to determine the mRNA species bound by IGF2BP1. RIP-enrichment of BCL11A mRNA was identified (4.6 ± 1.2 fold compared to input sample), as well as mRNA encoding other modifiers of globin gene expression including KLF1 and ZBTB7A. Further analyses of BCL11A showed no significant changes in BCL11A mRNA levels among IGF2BP1-OE cells (control: 6.5.E+03 ± 3.8.E+03, IGF2BP1-OE: 4.1.E+03 ± 1.2.E+02, p=0.403). However, BCL11A protein detection was reduced to background levels by Western analysis compared to transduction control cell lysates. These studies suggest that RNA-binding and cytoplasmic compartmentalization provide IGF2BP1 with a mechanism for increasing fetal hemoglobin via post-transcriptional regulation of globin gene transcription factors. Disclosures No relevant conflicts of interest to declare.

Description: Multiple components of the highly conserved let-7 cascade are developmentally regulated during the fetal-to adult transition of human ontogeny. Previous studies showed the let-7 miRNAs to be correlated with the developmentally regulated expression of fetal hemoglobin (HbF) in humans. Ex vivo manipulation of the main known let-7 regulator (LIN28) and downstream let-7 targets (IGF2BP1, IGF2BP3 and HMGA2) have shown variant increases in HbF levels (ranging from around 15-68% HbF) among cultured adult human erythroid cells. In addition, IGF2BP1 over-expression (OE) in cord blood erythroblasts raised HbF levels to an average of 90% compared to 55% in control transductions. Remarkably, in the fetal environment, the manipulation of the let-7 cascade has profound effects on HbF levels (cord blood control for the OE vectors: 57.4 ± 8.6%; LIN28A-OE: 90.8 ± 1.7%, p = 0.0003; IGF2BP3-OE: 79.0 ± 3.8%, p=0.001; HMGA2-OE: 73.4 ± 3.1, p=0.014; control for the tough decoy (TuD) vector: 51.6 ± 5.7%; let-7a-TuD: 90.3 ± 2.5%, p=0.001). As such, we hypothesized that the variable HbF effects in the adult cells may be due to independent or parallel effects from each component of the let-7 cascade since LIN28, IGF2BP1, IGF2BP3 and HMGA2 are expressed at very low levels or undetectable in the erythroid compartment after the fetal-to-adult transition. For these studies, lentiviral transduction of human CD34+ cells were investigated in erythropoietin-supplemented serum-free cultures for 21 days and compared to donor-matched control transductions. Based upon its important role in globin gene regulation, mRNA and protein expression patterns of the transcription factor BCL11A were investigated by RT-qPCR and Western blot at day 14 as a mechanism for HbF regulation by the let-7 cascade components. BCL11A mRNA was significantly down-regulated after LIN28A-OE (control: 4.1.E+03 ± 8.2.E+02 copies/ng, LIN28A-OE: 1.7.E+03 ± 1.2.E+03 copies/ng, p=0.04) and let-7a -TuD (control: 1.7E+03 ± 4.5E+02 copies/ng, let-7a-TuD: 4.3E+02 ± 1.8E+02 copies/ng, p=0.003), while no significant differences were observed after IGF2BP1-OE (control: 5.42.E+03 ± 1.77.E+03 copies/ng, IGF2BP1-OE: 4.38.E+03 ± 2.57.E+02 copies/ng, p=0.375), IGF2BP3-OE (control: 5.64.E+02 ± 2.68.E+02 copies/ng, IGF2BP3-OE: 6.70.E+02 ± 3.47.E+02 copies/ng, p=0.694) and HMGA2-OE (control: 4.26E+02 ± 8.18E+01 copies/ng, HMGA2-OE: 2.84E+02 ± 1.48E+02 copies/ng, p=0.104). Interestingly, the protein levels of BCL11A were consistently down-regulated only by LIN28A-OE, let-7a -TuD and IGF2BP1-OE, with minimal changes observed after IGF2BP3-OE and HMGA2-OE. To investigate which elements of the let-7 cascade are required for the fetal hemoglobin effects in adult cells, the expression levels of each component were compared with HbF effects after lentiviral-mediated transduction of LIN28A-OE, let-7a -TuD, IGF2BP1-OE, IGF2BP3-OE, or HMGA2-OE. RT-qPCR analyses were performed at day 14 and HPLC at day 21. HbF levels for the control transductions were less than 5%. Following LIN28A-OE (HbF: LIN28A-OE: 34.8 ± 2.7%, p=0.028), both IGF2BP1 and IGF2BP3 mRNA levels remained below the detection limits, while HMGA2 was slightly down-regulated. After let-7a -TuD (HbF: let-7a-TuD: 31.0 ± 5.0%, p=0.09), LIN28A and HMGA2 remained at background levels while IGF2BP1 and IGF2BP3 were below the detection limits. Following IGF2BP1-OE (HbF: IGF2BP1-OE: 64.6 ± 3.4%, p=0.02), LIN28A, IGF2BP3, and HMGA2 remained undetectable or at background levels. After IGF2BP3-OE (HbF: IGF2BP3-OE: 22.8 ± 4.4%, p=0.021), LIN28A, IGF2BP1 and HMGA2 also remained unchanged at background or undetectable levels. Finally, after HMGA2-OE (HbF: HMGA2-OE: 13.5 ± 1.5%, p=0.01), LIN28A, IGF2BP1 and IGF2BP3 remained below the detection limits. These results were confirmed by Western analysis, with the exception of HMGA2, which was consistently increased at the protein level following LIN28A-OE, let-7a -TuD, IGF2BP1-OE and IGF2BP3-OE. In summary, these results demonstrate that several components the let-7 cascade share a downstream mechanism of BCL11A protein suppression in human erythroblasts. However, the variability of BCL11A and HbF effects as well as the ability of LIN28A-OE, let-7a-TuD and HMGA2-OE to increase HbF in the absence of IGF2BP1 and IGF2BP3 suggest both shared and independent mechanisms for hemoglobin regulation by the let-7 cascade during the human fetal-to-adult switch. Disclosures No relevant conflicts of interest to declare.