Mitosis Is Not Required for Coordinated Changes in Iron Metabolism and High-Level Globin Gene Transcription in Erythropoietin-Responsive CD34+ Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1776-1776
Author(s):  
Ajoy V. Bhupatiraju ◽  
Nicole M. Josleyn ◽  
Y. Terry Lee ◽  
Jeffery L. Miller
Keyword(s):  
Cell Division ◽  
Globin Gene ◽  
Beta Globin ◽  
Globin Mrna ◽  
Gamma Globin ◽  
Cell Divisions ◽  
Adult Human ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Very High

Abstract Within the erythroid lineage, erythropoietin (EPO) responsiveness is manifested by cell division and growth as well as specific changes in heme and globin production that ultimately result in the production of erythrocytes. However, the nascent relationship between EPO-associated mitosis and globin gene regulation has not been fully defined. In this study, cultured adult human CD34+ cells from peripheral blood were used to investigate early cellular responses to erythropoietin in the context of mitosis. Matched cultures were performed in replicate using human cells from at least two healthy adults. To detect mitosis, one million cells were labeled with 2uM carboxyfluorescein diacetate succinamidyl ester (CFSE). The CFSE-labeled cells were then cultured in the presence [EPO(+)] or absence [EPO(−)] of 4U/mL EPO, and analyzed using flow cytometry. No cell divisions were detected in either condition during the first 24 hours in culture, and multiple cell divisions were noted on subsequent culture days only in the EPO(+) cultures. Remarkably, dual-staining with CFSE and CD71 revealed a small (<1%) population of the undivided cells at 24 hours with very high surface levels of transferrin receptor [CD71(++++)] exclusively in the EPO(+) cultures. Further analysis of those rare EPO-responsive, pre-mitotic cells revealed DNA synthesis and entry into the cell cycle in 62.5±4.7% compared with 1.5±1.8% among the cells with lower CD71 expression. None expressed glycophorin A. Based upon their distinct phenotype, single cells were sorted into 96-well plates, with sorting confirmation by quantitative RT-PCR of GAPDH mRNA (20 copies/cell detection limit). Next, gamma- and beta-globin transcripts were amplified for comparison (4 separate experiments). Among the pre-mitotic, CD71(++++) population, 20 of 182 total sorted cells (11%) lacked detectable levels of gamma- and beta-globin mRNA. Only 1 of 182 cells (0.5%) expressed gamma-globin mRNA and no detectable beta globin mRNA. 138 of 182 cells (76%) expressed only beta-globin mRNA, and 23 of 182 cells (13%) expressed both globin mRNAs. The median and mean levels of gamma-globin mRNA among the 24 gamma(+) cells were 92 and 245±1015 copies/cell respectively. In contrast, the median and mean levels of beta-globin mRNA among the 161 beta(+) cells were 1624 and 3999±5892 copies/cell respectively. By comparison, the CD34+ cells with low levels of surface CD71 in either EPO(+) or EPO(−) cultures demonstrated detectable levels of gamma globin mRNA in only 7 of 145 sorted cells (5%), and beta-globin mRNA in 85 of 145 cells (59%), with total (gamma+beta) globin mRNA under 200 copies/cell in >90% of those cells. These novel results suggest that the frequency and levels of gamma-globin transcripts are quite low at the earliest stages of an EPO response among adult human CD34+ cells. However, it is clearly demonstrated that the cells can increase their capacity to import iron and transcribe beta-globin mRNA at very high levels prior to their first EPO-dependent cell division.

scholarly journals Diagnosis of thalassemia using cDNA amplification of circulating erythroid cell mRNA with the polymerase chain reaction [published erratum appears in Blood 1992 Jun 15;79(12):3397]

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2433-2437 ◽  
Author(s):  
SZ Huang ◽  
GP Rodgers ◽  
FY Zeng ◽  
YT Zeng ◽  
AN Schechter
Keyword(s):  
Polymerase Chain Reaction ◽  
Globin Gene ◽  
Erythroid Cell ◽  
Mrna Levels ◽  
Beta Globin ◽  
Chain Reaction ◽  
Globin Mrna ◽  
Gamma Globin ◽  
Polymerase Chain ◽  
Alpha Beta

Abstract We have developed a technique to diagnose the alpha- and beta- thalassemia (thal) syndromes using the polymerase chain reaction to amplify cDNA copies of circulating erythroid cell messenger RNA (mRNA) so as to quantitate the relative amounts of alpha-, beta-, and gamma- globin mRNA contained therein. Quantitation, performed by scintillation counting of 32P-dCTP incorporated into specific globin cDNA bands, showed ratios of alpha/beta-globin mRNA greater than 10-fold and greater than fivefold increased in patients with beta 0- and beta (+)- thal, respectively, as well as a relative increase in gamma-globin mRNA levels. Conversely, patients with alpha-thalassemia showed a decreased ratio of alpha/beta-globin mRNA proportional to the number of alpha- globin genes deleted. This methodology of ascertaining ratios of globin mRNA species provides a new, simplified approach toward the diagnosis of thalassemia syndromes, and may be of value in other studies of globin gene expression at the transcription level.

Ineffective Erythropoiesis and Production of Normoblasts with a Beta Thalassemia Major Phenotype Using CD34+ Cells From Healthy Donors

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1085-1085
Author(s):  
Y.Terry Lee ◽  
Colleen Byrnes ◽  
Emily Riehm Meier ◽  
Antoinette Rabel ◽  
Jeffery L. Miller
Keyword(s):  
Ex Vivo ◽  
Globin Gene ◽  
Thalassemia Major ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Ineffective Erythropoiesis ◽  
Globin Mrna ◽  
Cell Counts ◽  
Cd34 Cells ◽  
Gene And Protein Expression

Abstract Abstract 1085 Reversal of anemia is the major target of thalassemia research, but studies of the molecular and cellular basis of the ineffective erythropoiesis of thalassemia are limited by access to donor progenitor cells. Here we demonstrate that thalassemic erythropoiesis may be recapitulated ex vivo by reducing the expression of hemoglobin in cultured CD34+ cells. Using lentiviral transduction of progenitor cells obtained from three healthy adult human donors, shRNA molecules were screened for their ability to reduce beta-globin gene and protein expression over 21 days in culture. Cells transduced with a scrambled vector served as donor-matched controls. Among the screened shRNA, one named HBB caused a consistent and significant reduction in beta-globin mRNA and protein. Beta-globin mRNA was reduced to levels <10% (p<0.001) compared to that of the controls (day14/21), while maintaining expression of gamma- and alpha-globin mRNA. HPLC was performed on an equivalent number of cells sampled on culture day 21 for hemoglobin type (HbA vs. HbF) and quantitation (area under each HPLC peak). The HbA peak was reduced by 96%, and there was a minor increase in the HbF peak (1.6 fold) after HBB transduction. Based upon these quantitative changes in hemoglobin, HbF represented 49.3±9.3% in the HBB transduced population compared with 2.9±0.7% (p<0.01) in controls. On culture day 14, there was no significant difference in glycophorin A (CD235), transferrin receptor (CD71), or cellular morphology despite the reduction in beta-globin mRNA. However, impaired terminal differentiation was detected by retainment of surface CD71 and a lack of enucleation during the third week of culture. Cell counts were lower in HBB transduced cells during the final stages of erythroid differentiation with a 61% (p=0.03) reduction in total cell counts by day 21 when compared to controls. Annexin V assay on day 21 also demonstrated increased phosphatidylserine expression in the HBB transduced cells [HBB=55.7±14.4% vs. Control=25.0±3.0%] in association with the decreased terminal differentiation. GDF15 quantitation demonstrated a significant (p=0.006) increase in the culture supernatants of HBB transduced cells. Sorted cytospin preparations revealed a distinct population of mature normoblasts containing a highly condensed nucleus surrounded by a thin ring of hypochromic cytoplasm. Reduction of erythroblast beta-globin gene and protein expression to levels associated with beta thalassemia major in humans causes ineffective erythropoiesis ex vivo by reducing cell production, increasing surface expression of phosphatidylserine, and impairing enucleation during terminal maturation. Efforts are now underway to use the culture system to explore mechanisms whereby reduced hemoglobin synthesis causes normoblast defects, and for screening of chemical and genetic rescue therapies for the thalassemic erythroid phenotype. Disclosures: No relevant conflicts of interest to declare.

scholarly journals IGF2BP1 Reverses Hemoglobin Switching in Adult Erythroblasts

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 639-639
Author(s):  
Laxminath Tumburu ◽  
Colleen Byrnes ◽  
Y. Terry Lee ◽  
Jaira F. de Vasconcellos ◽  
Antoinette Rabel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Adult Stage ◽  
Globin Gene ◽  
Expression Patterns ◽  
Beta Globin ◽  
Globin Mrna ◽  
Beta Globin Gene ◽  
Empty Vector ◽  
Cd34 Cells ◽  
Gene And Protein Expression

Abstract 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.

scholarly journals Fetal hemoglobin silencing in humans

Blood ◽  
2006 ◽  
Vol 108 (6) ◽  
pp. 2081-2086 ◽  
Author(s):  
Patricia A. Oneal ◽  
Nicole M. Gantt ◽  
Joseph D. Schwartz ◽  
Natarajan V. Bhanu ◽  
Y. Terry Lee ◽  
...  
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Postnatal Life ◽  
Globin Genes ◽  
Beta Globin ◽  
Globin Mrna ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
Age Related

Abstract Interruption of the normal fetal-to-adult transition of hemoglobin expression should largely ameliorate sickle cell and beta-thalassemia syndromes. Achievement of this clinical goal requires a robust understanding of gamma-globin gene and protein silencing during human development. For this purpose, age-related changes in globin phenotypes of circulating human erythroid cells were examined from 5 umbilical cords, 99 infants, and 5 adult donors. Unexpectedly, an average of 95% of the cord blood erythrocytes and reticulocytes expressed HbA and the adult beta-globin gene, as well as HbF and the gamma-globin genes. The distribution of hemoglobin and globin gene expression then changed abruptly due to the expansion of cells lacking HbF or gamma-globin mRNA (silenced cells). In adult reticulocytes, less than 5% expressed gamma-globin mRNA. These data are consistent with a “switching” model in humans that initially results largely from gamma- and beta-globin gene coexpression and competition during fetal development. In contrast, early postnatal life is marked by the rapid accumulation of cells that possess undetectable gamma-globin mRNA and HbF. The silencing phenomenon is mediated by a mechanism of cellular replacement. This novel silencing pattern may be important for the development of HbF-enhancing therapies.

Genetic Correction of Sickle Cell Disease by Co-Regulated γ-Globin Transgene Expression and ßS Interference.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1280-1280
Author(s):  
Selda Samakoglu ◽  
Yelena Usachenko ◽  
Tulin Budak-Alpdogan ◽  
Santina Acuto ◽  
Rosalba DiMarzo ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Branch Point ◽  
Transgene Expression ◽  
Globin Gene ◽  
Cell Disease ◽  
Globin Mrna ◽  
Tissue Specific ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract RNA interference (RNAi) is a promising therapeutic strategy, but its application to stem cell-based gene therapy for the treatment of congenital or acquired disorders will require highly specific gene silencing. To ensure co-expression of a therapeutic transgene and a small interfering RNA (siRNA), we hypothesized that a promoter-less small hairpin RNA (shRNA) embedded within an intron could yield siRNA in tissue-specific fashion and thus achieve regulated RNAi. We demonstrate here that γ-globin expression and erythroid-specific siRNA generation can be achieved in mammalian cells, including human CD34+ cells. The shRNA was encoded under the transcriptional control of the human β-globin promoter, a prototypic tissue-specific Pol II promoter, and positioned at two different sites in the second intron or in the 5′-UTR of a recombinant human γ-globin gene. Three different genes were targeted in mouse erythroleukemia (MEL) cells, green fluorescent protein (EGFP), human sickle β-globin (β S) and endogenous mouse β-gobin. When cloned immediately upstream of the branch point, the siRNA was efficiently generated without altering γ-globin mRNA expression and processing, suggesting that hairpin positioning near the branch point is not detrimental to RNA splicing. When cloned near the 5′-end of the intron, the siRNA was structurally impaired, and the γ-globin mRNA levels greatly diminished. This strong effect of shRNA positioning is consistent with a quality control pathway of gene transcription, whereby introns harboring dsRNA stem loops are degraded if splicing is altered. The strong induction of interferon type I genes associated with the latter position but not the former correlated with the formation of small shRNA degradation products. Positioning of the shRNA in the 5′-UTR did not induce major interferon responses but severely compromised γ-globin expression. To further validate these findings in a clinically relevant model, we engineered an RNAi lentiviral vector in which the human sickle β-globin specific (β S) siRNA is embedded the second intron of a recombinant γ-globin gene. Following transduction of CD34+ cells from patients with sickle cell disease, γ-globin transgene expression was induced upon erythroid differentiation concomitant with a dramatic decrease of the β S transcripts. These findings fully support the principle of synergistic gene delivery and lariat-encoded RNAi in human CD34+ cells, demonstrating the feasibility of using lariat-embedded siRNA to potentiate globin gene transfer by reducing competition from endogenous β S globin chains. Importantly, a moderate decrease in β S expression may substantially improve SCD and abrogate the need for high level expression of the vector-encoded globin gene. This approach to regulate RNAi may find broad applicability in a wide range of disorders where the concomitant expression of a transgene and RNAi will enhance treatment safety and/or efficacy.

scholarly journals Diagnosis of thalassemia using cDNA amplification of circulating erythroid cell mRNA with the polymerase chain reaction [published erratum appears in Blood 1992 Jun 15;79(12):3397]

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2433-2437
Author(s):  
SZ Huang ◽  
GP Rodgers ◽  
FY Zeng ◽  
YT Zeng ◽  
AN Schechter
Keyword(s):  
Polymerase Chain Reaction ◽  
Globin Gene ◽  
Erythroid Cell ◽  
Mrna Levels ◽  
Beta Globin ◽  
Chain Reaction ◽  
Globin Mrna ◽  
Gamma Globin ◽  
Polymerase Chain ◽  
Alpha Beta

We have developed a technique to diagnose the alpha- and beta- thalassemia (thal) syndromes using the polymerase chain reaction to amplify cDNA copies of circulating erythroid cell messenger RNA (mRNA) so as to quantitate the relative amounts of alpha-, beta-, and gamma- globin mRNA contained therein. Quantitation, performed by scintillation counting of 32P-dCTP incorporated into specific globin cDNA bands, showed ratios of alpha/beta-globin mRNA greater than 10-fold and greater than fivefold increased in patients with beta 0- and beta (+)- thal, respectively, as well as a relative increase in gamma-globin mRNA levels. Conversely, patients with alpha-thalassemia showed a decreased ratio of alpha/beta-globin mRNA proportional to the number of alpha- globin genes deleted. This methodology of ascertaining ratios of globin mRNA species provides a new, simplified approach toward the diagnosis of thalassemia syndromes, and may be of value in other studies of globin gene expression at the transcription level.

A sustained and pancellular reversal of gamma-globin gene silencing in adult human erythroid precursor cells

Blood ◽  
2005 ◽  
Vol 105 (1) ◽  
pp. 387-393 ◽  
Author(s):  
Natarajan V. Bhanu ◽  
Tiffany A. Trice ◽  
Y. Terry Lee ◽  
Nicole M. Gantt ◽  
Patricia Oneal ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Synergistic Effects ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Globin Mrna ◽  
Erythroid Precursor ◽  
Gamma Globin ◽  
Adult Human ◽  
Gene And Protein Expression ◽  
Complete Reversal

Abstract We systematically compared cytokine-mediated increases or decreases in proliferation with globin gene and protein expression in adult human erythroblasts. Despite their opposite effects on growth, stem cell factor (SCF) and transforming growth factorbeta (TGF-B) had synergistic effects with respect to fetal hemoglobin (HbF): average HbF/HbF + adult hemoglobin (HbA) ratio in erythropoietin (EPO) = 1.4 ± 1.0%; EPO + TGF-B = 10.8 ± 1.9%; EPO + SCF = 19.1 ± 6.2%; and EPO + SCF + TGF-B (EST) = 39.3 ± 6.3%. Polymerase chain reaction (PCR) revealed significant increases in gamma-globin transcripts that were balanced by reduced beta-globin transcripts. Single-cell quantitative PCR demonstrated a complete reversal of gamma-globin gene silencing with detectable gamma-globin mRNA in more than 95% of the cells. Immunostaining with HbF antibodies also showed a pancellular distribution in EST (96.2 ± 0.01% HbF positive) compared with a heterocellular distribution in EPO (42.9 ± 0.01% HbF positive). As shown here for the first time, a robust and pancellular reversal of gamma-globin gene silencing among hemoglobinized erythroblasts from adult humans may be achieved in the absence of hereditary mutation or direct genomic manipulation. (Blood. 2005;105:387-393)

Lentiviral Vector-Mediated Transfer of the Gamma-Globin Gene Into Normal and Beta-Thalassemic Human CD34+ Cells Results in Potentially Therapeutic Levels of Fetal Hemoglobin in Erythroid Progeny.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3579-3579
Author(s):  
Andrew Wilber ◽  
Phillip W. Hargrove ◽  
Yoon-Sang Kim ◽  
Arthur W. Nienhuis ◽  
Derek A. Persons
Keyword(s):  
Gene Transfer ◽  
Lentiviral Vector ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Glycophorin A ◽  
Beta Globin ◽  
Gamma Globin ◽  
Cd34 Cells ◽  
Chain Imbalance

Abstract Abstract 3579 Poster Board III-516 Beta-thalassemia results from severely reduced or absent expression of the beta-globin chain of hemoglobin, leading to severe anemia which is dependent upon transfusion for survival. The pathophysiology centers on the alpha- to beta-globin chain imbalance, which results in precipitation of excess alpha chains. These precipitates caused oxidative stress, membrane damage, and apoptosis of erythroid precursors in the bone marrow (BM). Allogeneic stem cell transplant can be curative but is only available to individuals with a matched donor. It is well known that increased levels of fetal hemoglobin (α2γ2;HbF), such as in the case of hereditary persistence of fetal hemoglobin, ameliorate the clinical severity of beta-thalassemia. We therefore developed a self-inactivating (SIN), erythroid-specific, gamma-globin lentiviral vector with the goal of reducing the chain imbalance through the production of HbF in red blood cells following transduction and transplantation of autologous HSCs. The vector, termed V5m3, contains 3.1-kb of transcriptional regulatory sequences from the β-globin locus control region, a 130-bp beta-globin promoter regulating transcription of the gamma-globin gene, and 3′ untranslated sequences from the native beta-globin gene. We previously showed that this vector was effective in correcting mouse models of beta-thalassemia and sickle cell disease (SCD). We further modified V5m3 to include a 400-bp chicken HS4 insulator element. Mobilized peripheral blood (PB)- or steady state bone marrow (BM)-derived CD34+ cells from normal or thalassemic human donors were used to evaluate vector performance. A two-phase in vitro model of human erythropoiesis was used in which a seed population of CD34+ cells is first expanded and then differentiated into late stage erythroblasts over a two week period. Gene transfer is performed 48 hours after initiation of culture. In this model, we routinely observe a 1000-fold expansion in total cell numbers where the vast majority of maturing erythroid cells are late stage erythroblasts reflected by nearly complete enrichment for expression of transferrin receptor (CD71; ≥98%) and glycophorin A (CD235; ≥80%) and loss or reduced expression of CD34 and CD45. Erythroid cells generated from normal adult PB and BM CD34+ cells demonstrate an adult pattern of hemoglobin production (HbA>96%;HbF<2%), as measured by acetate gel electrophoresis and HPLC, making this model ideal to evaluate enhancement of HbF levels by gene transfer. Normal PB CD34+ cells from four independent donors were transduced with the gamma-globin vector (MOI=20) or a GFP control vector (MOI=5). Vector transduction had no effect on cell growth or differentiation as monitored by consistent increases in total cell numbers and the appearance of CD71 (transferrin receptor) and glycophorin A on most cells (≥98% and '80%, respectively). The GFP vector achieved an average transduction rate of 87+/−6% and erythroblasts expressed low levels of HbF (1.7+/−0.6). Gamma globin gene transfer with the V5m3 (N=2) and V5m3-400 (N=4) vectors resulted in HbF levels ranging from 6 to 25%, with an average vector copy number of 0.8 to 1.1. We next tested the V5m3-400 vector using BM CD34+ cells from two patients with beta-thalassemia major. High levels of gene transfer were obtained with both the GFP and the globin vector, as evidenced by bulk marking (74+/−6% GFP+) or PCR analysis of CFU for presence of the V5m3-400 vector (12/12 positive Exp 1); (18/20 Exp 2); (18/24 Exp 3). Again, gene transfer did not perturb erythroid differentiation as monitored by the appearance glycophorin A (88+/−9%; GFP control and 86+/−6%; V5m3-400) as well as cell morphology. Erythroblasts derived from GFP transduced cells had a mean HbF level of 26+/−5% whereas those derived from cells transduced with V5m3-400 demonstrated a 100% increase of HbF to 58+/−2% with an average vector copy number of 0.6-0.8. Importantly, cultures of cells transduced with the globin vector demonstrated an average of 30% reduction in apoptotic cells (10% tunnel+) cells compared to GFP transduced control cells (15% tunnel+), suggesting rescue of erythroblasts through correction of the globin chain imbalance. Our data show that potentially therapeutic levels of HbF in thalassemic erythroblasts can be obtained following gene transfer using a gamma-globin lentiviral vector. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CD14+ monocytes repress gamma globin expression at early stages of erythropoiesis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Steven Heshusius ◽  
Esther Heideveld ◽  
Marieke von Lindern ◽  
Emile van den Akker
Keyword(s):  
Mononuclear Cells ◽  
Therapeutic Option ◽  
Globin Genes ◽  
Beta Globin ◽  
Hematopoietic Progenitors ◽  
Peripheral Blood Mononuclear ◽  
Temporal Regulation ◽  
Gamma Globin ◽  
Cd34 Cells ◽  
Adult Hemoglobin

AbstractIn β-hemoglobinopathies, reactivation of gamma- at the expense of beta-globin is a prominent therapeutic option. Expression of the globin genes is not strictly intrinsically regulated during erythropoiesis, supported by the observation that fetal erythroid cells switch to adult hemoglobin expression when injected in mice. We show cultured erythroblasts are a mix of HbA restrictive and HbA/HbF expressing cells and that the proportion of cells in the latter population depends on the starting material. Cultures started from CD34+ cells contain more HbA/HbF expressing cells compared to erythroblasts cultured from total peripheral blood mononuclear cells (PBMC). Depletion of CD14+ cells from PBMC resulted in higher HbF/HbA percentages. Conversely, CD34+ co-culture with CD14+ cells reduced the HbF/HbA population through cell–cell proximity, indicating that CD14+ actively repressed HbF expression in adult erythroid cultures. RNA-sequencing showed that HbA and HbA/HbF populations contain a limited number of differentially expressed genes, aside from HBG1/2. Co-culture of CD14+ cells with sorted uncommitted hematopoietic progenitors and CD34-CD36+ erythroblasts showed that hematopoietic progenitors prior to the hemoglobinized erythroid stages are more readily influenced by CD14+ cells to downregulate expression of HBG1/2, suggesting temporal regulation of these genes. This possibly provides a novel therapeutic avenue to develop β-hemoglobinopathies treatments.

scholarly journals Serum factors can modulate the developmental clock of gamma- to beta-globin gene switching in somatic cell hybrids.

1993 ◽  
Vol 13 (8) ◽  
pp. 4844-4851 ◽  
Author(s):  
G Zitnik ◽  
Q Li ◽  
G Stamatoyannopoulos ◽  
T Papayannopoulou
Keyword(s):  
Globin Gene ◽  
Cumulative Number ◽  
Beta Globin ◽  
Serum Factors ◽  
Beta Globin Gene ◽  
Cell Divisions ◽  
Growth Promoting ◽  
Gene Switching ◽  
Mouse Erythroleukemia

The fusion of human fetal erythroid (HFE) cells with mouse erythroleukemia (MEL) cells produces stable synkaryons (HFE x MEL) which can be monitored for extended periods of time in culture. Initially these hybrids express a human fetal globin program (gamma >> beta), but after weeks or months in culture, they switch to an adult pattern of globin expression (beta >> gamma). The rate at which hybrids switch to the adult phenotype is roughly dependent on the gestational age of the fetal erythroid cells used in the fusion, suggesting that the rate of switching in vitro may be determined by a developmental clock type of mechanism, possibly involving the cumulative number of divisions experienced by the human fetal cells. To investigate whether the number or rate of cell divisions postfusion can influence the rate of switching, we monitored the rate of switching in hybrids from independent fusions under growth-promoting (serum-replete) and growth-suppressing (serum-deprived) conditions. We found that hybrids grown under serum-deprived or serumless conditions switched more rapidly to adult globin expression than did their counterparts in serum-replete conditions. Neither the number of cumulative cell divisions nor time in culture per se predicted the rate of switching in vitro. Our data suggest that factors present in serum either retard switching of hybrids by their presence or promote switching by their absence, indicating that globin switching in vitro can be modulated by the environment; however, once switching in HFE x MEL hybrids is complete, serum factors cannot reverse this process.

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