scholarly journals Combining HPFH Mutations in Human Adult HSCs to Enhance Reactivation of Fetal Hemoglobin

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2246-2246
Author(s):  
Nikoletta Psatha ◽  
Afrodite Georgakopoulou ◽  
Chang Li ◽  
Pavel Sova ◽  
Hongjie Wang ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Fetal Hemoglobin ◽  
Beta Globin ◽  
Gamma Globin ◽  
Hbf Induction ◽  
Hereditary Persistence ◽  
Globin Promoter ◽  
Cis And Trans

Despite the significant advances in developing novel therapies for hemoglobinopathies, developing a universal and robust therapeutic approach, able to achieve an event-free result in all β0/β0 thalassemic and sickle cell patients is a work in progress. It is known that increase of Fetal hemoglobin (HbF) can lead to an ameliorated clinical picture up to transfusion independency. Inactivation via genome editing of γ-globin suppressors or introduction of Hereditary Persistence of Fetal Hemoglobin (HPFH) mutations in the promoter of hemoglobin gamma (HBG) have been shown to significantly increase the endogenous HbF expression. Here we studied the effect in HbF reactivation by targeting the binding site of the two main HbF silencers, BCL11a and LRF and how it compares to the well-established editing of the BCL11a erythroid enhancer. Furthermore, in order to achieve higher levels of fetal hemoglobin, we assayed the effect of all combinations of these cis and trans acting mutations. We first observed that all single knock outs had similar effects in HbF reactivation. Interestingly, by targeting both sites in the gamma globin promoter we observed a decrease in HbF induction compared to each single edit, suggesting a possible binding of a gamma globin activator in the in-between region. However, editing of the BCL11a erythroid enhancer and either of the two silencer binding sites yielded maximum levels of fetal hemoglobin, with pancellular HbF expression, both in vitro and in vivo following xeno-transplantation in NSGW mice. Collectively, our data suggest that the combination of two fetal globin reactivation mutations, one cis and one trans, have the potential to significantly increase HbF both totally and on per cell basis. This strategy has the potential to induce higher levels of HbF reactivation with a clinical benefit in patients with beta globin disorders. Disclosures No relevant conflicts of interest to declare.

scholarly journals The beta-globin gene on the Chinese delta beta-thalassemia chromosome carries a promoter mutation

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1470-1474 ◽  
Author(s):  
GF Atweh ◽  
XX Zhu ◽  
HE Brickner ◽  
CH Dowling ◽  
HH Jr Kazazian ◽  
...  
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Promoter Mutation ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
New Type

A new type of delta beta-thalassemia characterized by decreased expression of the beta-globin gene and increased expression of both G gamma and A gamma globin gene in the absence of a detectable deletion has recently been described in the Chinese population. In this study we characterize the mutant beta-globin gene from this delta beta- thalassemia chromosome. An A to G transversion is identified in the “ATA” sequence of the promoter region that leads to decreased expression of the beta-globin gene in vivo and in vitro. We also demonstrate the presence of this mutation in every individual with a high fetal hemoglobin phenotype in this family and its absence in every individual with a normal hemoglobin phenotype. This same promoter mutation has recently been detected in Chinese beta-thalassemia genes where it is present on chromosomes of the same haplotype as that of the delta beta-thalassemia chromosome we are studying. These data support the hypothesis that an as yet unidentified mutation occurred on the ancestral chromosome carrying the promoter mutation and subsequently gave rise to the delta beta-thalassemia phenotype.

scholarly journals The beta-globin gene on the Chinese delta beta-thalassemia chromosome carries a promoter mutation

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1470-1474 ◽  
Author(s):  
GF Atweh ◽  
XX Zhu ◽  
HE Brickner ◽  
CH Dowling ◽  
HH Jr Kazazian ◽  
...  
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Promoter Mutation ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
New Type

Abstract A new type of delta beta-thalassemia characterized by decreased expression of the beta-globin gene and increased expression of both G gamma and A gamma globin gene in the absence of a detectable deletion has recently been described in the Chinese population. In this study we characterize the mutant beta-globin gene from this delta beta- thalassemia chromosome. An A to G transversion is identified in the “ATA” sequence of the promoter region that leads to decreased expression of the beta-globin gene in vivo and in vitro. We also demonstrate the presence of this mutation in every individual with a high fetal hemoglobin phenotype in this family and its absence in every individual with a normal hemoglobin phenotype. This same promoter mutation has recently been detected in Chinese beta-thalassemia genes where it is present on chromosomes of the same haplotype as that of the delta beta-thalassemia chromosome we are studying. These data support the hypothesis that an as yet unidentified mutation occurred on the ancestral chromosome carrying the promoter mutation and subsequently gave rise to the delta beta-thalassemia phenotype.

scholarly journals Fetal hemoglobin induction by acetate, a product of butyrate catabolism [see comments]

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 3198-3204 ◽  
Author(s):  
G Stamatoyannopoulos ◽  
CA Blau ◽  
B Nakamoto ◽  
B Josephson ◽  
Q Li ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Sickle Cell Disease ◽  
Umbilical Cord Blood ◽  
Sodium Acetate ◽  
Fetal Hemoglobin ◽  
Cell Disease ◽  
Gamma Globin ◽  
Hbf Induction ◽  
Globin Synthesis

Abstract Butyrate induces fetal hemoglobin (HbF) synthesis in cultures of erythroid progenitors, in primates, and in man. The mechanism by which this compound stimulates gamma-globin synthesis is unknown. In the course of butyrate catabolism, beta oxidation by mitochondrial enzymes results in the formation of two acetate molecules from each molecule of butyrate. Studies were performed to determine whether acetate itself induces HbF synthesis. In erythroid burst-forming unit (BFU-E) cultures from normal persons, and individuals with sickle cell disease and umbilical-cord blood, dose-dependent increases in gamma-globin protein and gamma mRNA were consistently observed in response to increasing acetate concentrations. In BFU-E cultures from normal adults and patients with sickle cell disease, the ratio of gamma/gamma + beta mRNA increased twofold to fivefold in response to acetate, whereas the percentage of BFU-E progeny staining with an anti-gamma monoclonal antibody (MoAb) increased approximately twofold. Acetate-induced increases in gamma-gene expression were also noted in the progeny of umbilical cord blood BFU-E, although the magnitude of change in response to acetate was less because of a higher baseline of gamma- chain production. The effect of acetate on HbF induction in vivo was evaluated using transgenic mouse and primate models. A transgenic mouse bearing a 2.5-kb mu locus control region (mu LCR) cassette linked to a 3.3-kb A gamma gene displayed a near twofold increase in gamma mRNA during a 10-day infusion of sodium acetate at a dose of 1.5 g/kg/d. Sodium acetate administration in baboons, in doses ranging from 1.5 to 6 g/kg/d by continuous intravenous infusion, also resulted in the stimulation of gamma-globin synthesis, with the percentage of HbF- containing reticulocytes (F reticulocytes) approaching 30%. Surprisingly, a dose-response effect of acetate on HbF induction was not observed in the baboons, and HbF induction was not sustained with prolonged acetate administration. These results suggest that both two- carbon fatty acids (acetate) and four-carbon fatty acids (butyrate) stimulate synthesis of HbF in vivo.

scholarly journals Truncated Erythropoietin Receptors Confer an In Vivo Selective Advantage in Gene-Modified Erythroid Cells Expressing Fetal Hemoglobin Due to BCL11A Interference

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2063-2063
Author(s):  
Naoya Uchida ◽  
Claire Drysdale ◽  
Morgan Yapundich ◽  
Jackson Gamer ◽  
Tina Nassehi ◽  
...  
Keyword(s):  
Rhesus Macaques ◽  
Fetal Hemoglobin ◽  
Selective Advantage ◽  
Erythroid Cells ◽  
Post Transplant ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Hbf Induction

Hematopoietic stem cell gene therapy for hemoglobin disorders, such as sickle cell disease, requires high-level gene marking and robust therapeutic globin expression in erythroid cells (>20% of γ- or β-globin production) for widespread successful clinical application. We previously demonstrated that lentiviral transduction of a truncated human erythropoietin receptor (thEpoR) gene allows for erythropoietin-dependent selective proliferation of gene-modified human erythroid cells during in vitro differentiation (ASH 2017). In this study, we sought to evaluate whether thEpoR can enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous non-human primate transplantation models. To investigate this hypothesis, we designed lentiviral vectors encoding both thEpoR and BCL11A-targeting micro RNA-adapted short hairpin RNA (shmiBCL11A), driven off an erythroid specific ankyrin 1 (ANK1) promoter. Both selective proliferation and high-level fetal hemoglobin (HbF) induction were observed in in vitro erythroid differentiation cultures using transduced human CD34+ cells. Healthy donor CD34+ cells were transduced with shmiBCL11A vector, thEpoR-shmiBCL11A vector, and GFP vector (control). Transduced cells were transplanted into immunodeficient NBSGW mice. Five months post-transplant, xenograft bone marrow cells were evaluated for human cell engraftment (human CD45+) and vector copy number (VCN) in both human CD34+ progenitor cells and glycophorin A+ (GPA+) erythroid cells. HbF production was also measured in GPA+ erythroid cells by reverse phase HPLC. We observed efficient transduction in transduced CD34+ cells in vitro (VCN 2.1-5.1) and similar human cell engraftment among all groups (84-89%). The VCN with thEpoR-shmiBCL11A transduction was 3-fold higher in human erythroid cells when compared to CD34+ cells (p<0.01), but not with shmiBCL11A or GFP vectors. HbF levels were significantly elevated in thEpoR-shmiBCL11A vector (43±6%, p<0.01) when compared to no transduction control (1±0%), but not for either shmiBCL11A vector (3±1%) or GFP vector (1±0%). These data demonstrate selective proliferation of gene-modified erythroid cells, as well as enhanced HbF induction with thEpoR-shmiBCL11A transduction. We then performed autologous rhesus CD34+ cell transplantation using either shmiBCL11A vector (142562 and RA0706, n=2, compared to a GPA promoter-derived shmiBCL11A vector) or thEpoR-shmiBCL11A vector (ZL50 and ZM24, n=2, compared to a Venus-encoding vector). Transduced CD34+ cells were transplanted into autologous rhesus macaques following 2x5Gy total body irradiation. Efficient transduction was observed in CD34+ cells in vitro among all 4 macaques (VCN 3.8-8.7) using a high-density culture protocol (Uchida N, Mol Ther Methods Clin Dev. 2019). In shmiBCL11A transduction animals, engraftment of gene-modified cells (VCN 0.2-1.0) and robust HbF induction (14-16%) were observed 1 month post-transplant. However, VCN and HbF levels were reduced down to VCN ~0.1 and HbF ~0.4% in both animals 6 months post-transplant. In contrast, a thEpoR-shmiBCL11A transduction animal (ZL50) resulted in engraftment of gene-modified cells (VCN 0.8-1.0) and robust HbF induction (~18%) 1 month post-transplant, with both gene marking and HbF levels remaining high at VCN 0.6-0.7 and HbF ~15% 4 months post-transplant. These data suggest that shmiBCL11A transduction results in transient HbF induction in gene-modified erythroid cells, while thEpoR-based selective advantage allows for sustained HbF induction with shmiBCL11A. In summary, we developed erythroid-specific thEpoR-shmiBCL11A expressing vectors, enhancing HbF induction in gene-modified erythroid cells in xenograft mice and rhesus macaques. While further in vivo studies are desirable, the use of thEpoR appears to provide a selective advantage for gene-modified erythroid cells in gene therapy strategies for hemoglobin disorders. Disclosures No relevant conflicts of interest to declare.

scholarly journals On the induction of fetal hemoglobin by butyrates: in vivo and in vitro studies with sodium butyrate and comparison of combination treatments with 5-AzaC and AraC

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 1963-1971 ◽  
Author(s):  
P Constantoulakis ◽  
G Knitter ◽  
G Stamatoyannopoulos
Keyword(s):  
Sodium Butyrate ◽  
Progenitor Cell ◽  
Fetal Hemoglobin ◽  
Globin Genes ◽  
Cell Numbers ◽  
Gamma Globin ◽  
Combination Treatments ◽  
Bone Marrow Cultures

Abstract To obtain information on the cellular mechanism of induction of fetal hemoglobin (HbF) by sodium butyrate (NaB), we treated adult baboons with NaB and assessed its effects on HbF expression. Infusion of NaB increased F reticulocytes and F-positive CFUe and e-cluster colonies without induction of reticulocytosis or increase in progenitor cell numbers. Addition of NaB in bone marrow cultures increased the frequency of F-positive CFUe and e-clusters without increasing progenitor cell numbers. NaB induced HbF in human adult BFUe cultures and increased the gamma/gamma + beta globin chain and mRNA ratios in short-term incubations of culture-derived erythroblasts. There was a synergistic induction of HbF by NaB and 5-azacytidine (5-azaC), but not when the animal was treated with NaB and cytarabine (AraC). Our results suggest that the activation of gamma-globin expression by NaB reflects an action of this compound on globin genes or globin chromatin.

scholarly journals A novel deletion of approximately 27 kb including the beta-globin gene and the locus control region 3'HS-1 regulatory sequence: beta zero- thalassemia or hereditary persistence of fetal hemoglobin?

Blood ◽  
1994 ◽  
Vol 83 (3) ◽  
pp. 822-827 ◽  
Author(s):  
AJ Dimovski ◽  
V Divoky ◽  
AD Adekile ◽  
E Baysal ◽  
JB Wilson ◽  
...  
Keyword(s):  
Control Region ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Locus Control Region ◽  
Regulatory Sequence ◽  
Beta Globin ◽  
Gamma Chain ◽  
Beta Globin Gene ◽  
Gamma Globin

Abstract A novel deletion of approximately 27 kb with the 5′ breakpoint 1.5 to 2.2 kb upstream of the beta-globin gene, and the 3′ breakpoint approximately 24 kb downstream of the beta-globin gene, has been found in five members of two families from Southeast Asia (Vietnam and Cambodia). Six members of another family from China, previously reported from our laboratory, have also been shown to carry this deletion. The patients presented with mild hypochromia and microcytosis, a hemoglobin (Hb) A2 level of approximately 4.0%, and a markedly increased, heterocellularly distributed, Hb F level (14.0 to 26.0%). In vitro globin-chain synthesis showed a mild imbalance with appreciable gamma-chain compensation (alpha/beta + gamma ratio of 1.46). The 3′ end of this deletion includes the 3′HS-1, and we hypothesize that removal of this region results in the loss of its gamma-globin gene-silencing effect, which causes a markedly elevated Hb F level with a modest increase in Hb A2 levels, unlike the situation in other deletional beta zero-thalassemias. The possible influence of particular sequence variations in the locus control region 5′HS-2 and the G gamma promoter, present on the chromosome with this deletion, on the overall gamma-globin gene should also be considered.

scholarly journals Sardinian G gamma-HPFH: a T----C substitution in a conserved "octamer" sequence in the G gamma-globin promoter

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 815-817 ◽  
Author(s):  
S Ottolenghi ◽  
S Nicolis ◽  
R Taramelli ◽  
N Malgaretti ◽  
R Mantovani ◽  
...  
Keyword(s):  
Globin Gene ◽  
Single Point ◽  
Point Mutations ◽  
Fetal Hemoglobin ◽  
Regulatory Elements ◽  
Globin Genes ◽  
Gamma Globin ◽  
New Type ◽  
Hereditary Persistence ◽  
Globin Promoter

Abstract A survey of hemoglobinopathies in Northern Sardinia allowed the identification of two subjects heterozygous for a new type of G gamma hereditary persistence of fetal hemoglobin (HPFH). The G gamma-globin gene from the HPFH chromosome shows the presence of a T----C substitution 175 nucleotides upstream of the CAP site, adding a new example of single-point mutations occurring in the promoter region of the gamma-globin genes and linked to HPFH phenotypes. In this case the mutation affects the 3′ end nucleotide of a conserved octamer sequence known to be present in other regulatory elements of several genes.

scholarly journals Fetal hemoglobin synthesis in erythroid cultures in hereditary persistence of fetal hemoglobin and beta o-thalassemia

Blood ◽  
1984 ◽  
Vol 63 (6) ◽  
pp. 1278-1284 ◽  
Author(s):  
RS Weinberg ◽  
SE Antonarakis ◽  
HH Jr Kazazian ◽  
GJ Dover ◽  
SH Orkin ◽  
...  
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Globin ◽  
Globin Chains ◽  
Hereditary Persistence ◽  
Enzyme Pattern ◽  
Globin Synthesis ◽  
The Individual ◽  
Globin Gene Expression

To determine whether hemoglobin regulation is normal in diseases affecting beta-globin gene expression, globin synthesis was examined in members of a family of a patient with hereditary persistence of fetal hemoglobin/beta o-thalassemia (HPFH/beta o-thal). The HPFH defect is the Ghanian type II, with a deletion from psi beta 1 to at least 20 kb 3′ to beta. The beta o-thal gene has the haplotype II restriction enzyme pattern and has the beta 39 nonsense mutation. Erythroid colonies from blood BFU-E were radiolabeled, and globin chains were separated by gel electrophoresis. Colonies from the beta o-thal heterozygote had non-alpha/alpha ratios more balanced than in the reticulocytes. Gamma synthesis was 11% of non-alpha, which is higher than in reticulocytes, but within the range seen in normal adult colonies. Both HPFH heterozygotes produced 20%-30% gamma in erythroid colonies as well as reticulocytes, although non-alpha/alpha was more balanced in the colonies. The HPFH/beta o-thal patient produced 100% gamma in reticulocytes and in colonies. G gamma and gamma-synthetic proportions were not correlated at the individual colony level in the heterozygotes, suggesting that they had “adult” and not “fetal” progenitor cells. The Hb expression of these adult progenitors is presumably modulated normally in vivo in beta o-thal, but the normal decrease in HbF production does not occur in gene deletion HPFH.

scholarly journals Fetal hemoglobin synthesis in erythroid cultures in hereditary persistence of fetal hemoglobin and beta o-thalassemia

Blood ◽  
1984 ◽  
Vol 63 (6) ◽  
pp. 1278-1284 ◽  
Author(s):  
RS Weinberg ◽  
SE Antonarakis ◽  
HH Jr Kazazian ◽  
GJ Dover ◽  
SH Orkin ◽  
...  
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Globin ◽  
Globin Chains ◽  
Hereditary Persistence ◽  
Enzyme Pattern ◽  
Globin Synthesis ◽  
The Individual ◽  
Globin Gene Expression

Abstract To determine whether hemoglobin regulation is normal in diseases affecting beta-globin gene expression, globin synthesis was examined in members of a family of a patient with hereditary persistence of fetal hemoglobin/beta o-thalassemia (HPFH/beta o-thal). The HPFH defect is the Ghanian type II, with a deletion from psi beta 1 to at least 20 kb 3′ to beta. The beta o-thal gene has the haplotype II restriction enzyme pattern and has the beta 39 nonsense mutation. Erythroid colonies from blood BFU-E were radiolabeled, and globin chains were separated by gel electrophoresis. Colonies from the beta o-thal heterozygote had non-alpha/alpha ratios more balanced than in the reticulocytes. Gamma synthesis was 11% of non-alpha, which is higher than in reticulocytes, but within the range seen in normal adult colonies. Both HPFH heterozygotes produced 20%-30% gamma in erythroid colonies as well as reticulocytes, although non-alpha/alpha was more balanced in the colonies. The HPFH/beta o-thal patient produced 100% gamma in reticulocytes and in colonies. G gamma and gamma-synthetic proportions were not correlated at the individual colony level in the heterozygotes, suggesting that they had “adult” and not “fetal” progenitor cells. The Hb expression of these adult progenitors is presumably modulated normally in vivo in beta o-thal, but the normal decrease in HbF production does not occur in gene deletion HPFH.

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