Analysis of Fetal Hemoglobin Expression within Humanized Sickle Cell Disease Mice Overexpressing the TR2/4 Transgene.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1619-1619 ◽  
Author(s):  
Andrew Campbell ◽  
Osamu Tanabe ◽  
Rebekah Urbonya ◽  
Andrea Mathias ◽  
Lihong Shi ◽  
...  
Keyword(s):  
Body Weight ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Globin ◽  
Cell Disease ◽  
Hemoglobin F ◽  
Gamma Globin ◽  
Fold Induction

Abstract Abstract 1619 Background: Sickle Cell Disease (SCD) is a chronic debilitating hematologic condition caused by a missense mutation within the adult beta globin gene leading to significant morbidity and mortality. Increased Fetal Hemoglobin production has been shown to significantly ameliorate SCD symptoms and improve survival. A novel specific DNA-binding factor DRED (direct repeat erythroid definitive) was recently identified that regulated epsilon and gamma globin gene expression (Tanimoto et al Genes Dev 2000). Purification of DRED revealed that it harbored the nuclear orphan hormone receptors, TR2/TR4, as its DNA binding core (Tanabe et al EMBO 2002). Overexpression of TR2/TR4 Transgene within Human Beta Globin Yeast Artificial Chromosome Transgenic Mice resulted in 4-fold induction of the gamma globin mRNA levels (Tanabe et al EMBO 2007). Therefore, we wanted to determine if the overexpression of TR2/TR4 within a humanized sickle cell disease model would result in fetal hemoglobin induction. Methods: Humanized Homozygous Knock-In UAB-Sickle Cell (UAB-Hbahα/hα Hbbhβs/hβs) Mice (Wu et al Blood 2006) was mated to TR2/TR4 Overexpressing Mice (TgTR2/TR4) to generate homozygous SS-TR2/TR4 compound heterozygotes (UAB-Hba hα/hα Hbb hβs/hβs TgTR2/TR4). We generated four 2–3 month old homozygous SS-TR2/TR4 transgenic mice and compared hemoglobin F levels, complete blood cell counts and % body weight (liver, spleen, kidney) to six 2–3 month old homozygous SS mice (Hbahα/hα Hbb hβs/hβs)without the overexpressing TgTR2/TR4. Tail PCR genotyping of all sickle cell mice (with and without TgTR2/TR4) and Hemoglobin F(Hgb F) and Sickle (HgbS) levels were confirmed by HPLC Hemoglobin electrophoresis. Results: The mean Hgb F: 7.8% (n=6, sd 1.63+/−) in the homozygous SS control mice vs. 16.5% (n=4, sd 2.64+/−)in the homozygous SS-TR2/4 Mice (2 Fold higher). Hematologic profile revealed a mean Hct: 25.2 (n=6, sd 5.50 +/−) mean MCV: 75.4 (n=6, sd 10+/−) and a mean WBC: 22.6 (n= 6, sd 13.9 +/−) in the homozygous SS control mice vs. a mean Hct: 31.25(n=4, sd 6.89+/−), mean MCV: 61(n=4, sd 3.5+/−) mean WBC: 16.3(n= 4, sd 5.99+/−) in the homozygous SS-TR2/TR4 mice. Lastly, initial organ (spleen, liver, kidney) pathology evaluation revealed decreased % body weight (bw) in homozygous SS TR2/TR4 Mice vs. homozygous SS controls: 1) Spleen %bw: 4.3% vs. 3.5% TgTR2/TR4), 2) Liver % bw: 8.8% vs. 7.7% TgTR2/TR4), and 3) Kidney %bw: 1.14% vs. 1.02% TgTR2/TR4). Conclusions: Our preliminary analysis revealed that TR2/TR4 overexpression within a humanized sickle cell disease mouse model resulted in a 2-fold induction of fetal hemoglobin based on HPLC hemoglobin electrophoresis. Further, increased TR2/TR4 overexpression improved anemia and organomegaly within sickle cell disease mice. TR2/TR4 may be an attractive target for fetal hemoglobin induction for the treatment of sickle cell disease. Ongoing studies will determine if TR2/TR4 decreases organ specific disease pathology. We will also determine the cellular distribution of fetal hemoglobin in future studies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Complementary Base Editing Approaches for the Treatment of Sickle Cell Disease and Beta Thalassemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3352-3352 ◽  
Author(s):  
Ling Lin ◽  
Adrian P. Rybak ◽  
Conrad Rinaldi ◽  
Jonathan Yen ◽  
Yanfang Fu ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Cell Disease ◽  
Hematopoietic Stem ◽  
Base Editing ◽  
Gamma Globin

Sickle cell disease (SCD) and Beta thalassemia are disorders of beta globin production and function that lead to severe anemia and significant disease complications across a multitude of organ systems. Autologous transplantation of hematopoietic stem cells engineered through the upregulation of fetal hemoglobin (HbF) or correction of the beta globin gene have the potential to reduce disease burden in patients with beta hemoglobinopathies. Base editing is a recently developed technology that enables precise modification of the genome without the introduction of double strand DNA breaks. Gamma globin gene promoters were comprehensively screened with cytosine and adenine base editors (ABE) for the identification of alterations that would derepress HbF. Three regions were identified that significantly upregulated HbF, and the most effective nucleotide residue conversions are supported by natural variation seen in patients with hereditary persistence of fetal hemoglobin (HPFH). ABEs have been developed that significantly increase the level of HbF following nucleotide conversion at key regulatory motifs within the HBG1 and HBG2 promoters. CD34+ hematopoietic stem and progenitor cells (HSPC) were purified at clinical scale and edited using a process designed to preserve self-renewal capacity. Editing at two independent sites with different ABEs reached 94 percent and resulted in up to 63 percent gamma globin by UPLC. The levels of HbF observed should afford protection to the majority of SCD and Beta thalassemia patients based on clinical observations of HPFH and non-interventional therapy that links higher HbF dosage with milder disease (Ngo et al, 2011 Brit J Hem; Musallam et al, 2012 Blood). Directly correcting the Glu6Val mutation of SCD has been a recent goal of genetic therapies designed for the SCD population. Current base editing technology cannot yet convert mutations like those that result from the A-T transversion in sickle beta globin; however, ABE variants have been designed to recognize and edit the opposite stranded adenine residue of valine. This results in the conversion of valine to alanine and the production of a naturally occurring variant known as Hb G-Makassar. Beta globin with alanine at this position does not contribute to polymer formation, and patients with Hb G-Makassar present with normal hematological parameters and red blood cell morphology. SCD patient fibroblasts edited with these ABE variants achieve up to 70 percent conversion of the target adenine. CD34 cells from healthy donors were then edited with a lead ABE variant, targeting a synonymous mutation in an adjacent proline that resides within the editing window and serves as a proxy for editing the SCD mutation. The average editing frequency was 40 percent. Donor myeloid chimerism documented at these levels in the allogeneic transplant setting exceeds the 20 percent that is required for reversing the sickle phenotype (Fitzhugh et al, 2017 Blood). These next generation editing approaches provide a promising new modality for treating patients with Beta thalassemia and SCD. Disclosures No relevant conflicts of interest to declare.

scholarly journals Analysis of hemoglobin F production in Saudi Arabian families with sickle cell anemia

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 716-720 ◽  
Author(s):  
BA Miller ◽  
M Salameh ◽  
M Ahmed ◽  
N Olivieri ◽  
G Antognetti ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Cell Disease ◽  
Erythroid Progenitors ◽  
Hemoglobin F ◽  
Gamma Globin ◽  
Hemoglobin Production

Erythrocytes and progenitor-derived erythroblasts of sickle cell anemia patients from the Eastern Province of Saudi Arabia contain increased fetal hemoglobin and G gamma globin. A distinctive DNA polymorphism haplotype in the beta globin gene cluster (++- +-), tightly coupled to a C----T substitution at position -158 5′ to the cap site of the G gamma globin gene, is strongly associated with sickle cell disease in this region. To determine whether the increased fetal hemoglobin production and/or elevated G gamma globin content are tightly linked to this haplotype, we studied 55 members of five Saudi families in which sickle cell disease is present. The results did not suggest a tight linkage of the haplotype to increased fetal hemoglobin production. On the other hand, several sickle trait family members heterozygous for the haplotype had normal fetal hemoglobin production in culture but elevated G gamma to A gamma ratios in peripheral blood. This observation suggests that in this genetic background increased expression of the G gamma globin gene may occur without a measurable increase in total fetal hemoglobin production. The family studies also clearly demonstrate that increased fetal hemoglobin production by erythroid progenitors is dependent on zygosity for the sickle gene in this population. These findings strongly suggest that other factors, such as the products of genes stimulated by hemolytic stress or other genetic determinants associated with the Saudi beta S chromosome, may interact with the -158 C----T substitution and influence gamma globin gene expression in this population.

scholarly journals A potential regulatory region for the expression of fetal hemoglobin in sickle cell disease

Blood ◽  
1994 ◽  
Vol 84 (1) ◽  
pp. 331-338
Author(s):  
S Pissard ◽  
Y Beuzard
Keyword(s):  
Sickle Cell Disease ◽  
Protein Binding ◽  
Sickle Cell ◽  
Globin Gene ◽  
Regulatory Region ◽  
Fetal Hemoglobin ◽  
Beta Globin ◽  
Cell Disease ◽  
Hemoglobin F ◽  
Binding Motifs

We describe a 0.5-kb region located 1.65 to 1.15 kb upstream of the G gamma fetal globin gene with three polymorphisms of erythroid and ubiquitous nuclear protein binding motifs (GATA, CRE, and a new protein binding site). These three polymorphisms result in high-affinity and low-affinity motifs for nuclear proteins, and are combined in four arrangements called pre-G gamma frameworks (pG gamma Fs). Each pG gamma F is linked with one of the major haplotypes of the beta-globin gene cluster observed in sickle cell disease (SCD) associated with different mean levels of hemoglobin F (Hb F) expression (P < .001). This strong linkage and the differing affinities suggest that this region may be involved in the modulation of Hb F expression in SCD.

scholarly journals Analysis of hemoglobin F production in Saudi Arabian families with sickle cell anemia

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 716-720 ◽  
Author(s):  
BA Miller ◽  
M Salameh ◽  
M Ahmed ◽  
N Olivieri ◽  
G Antognetti ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Cell Disease ◽  
Erythroid Progenitors ◽  
Hemoglobin F ◽  
Gamma Globin ◽  
Hemoglobin Production

Abstract Erythrocytes and progenitor-derived erythroblasts of sickle cell anemia patients from the Eastern Province of Saudi Arabia contain increased fetal hemoglobin and G gamma globin. A distinctive DNA polymorphism haplotype in the beta globin gene cluster (++- +-), tightly coupled to a C----T substitution at position -158 5′ to the cap site of the G gamma globin gene, is strongly associated with sickle cell disease in this region. To determine whether the increased fetal hemoglobin production and/or elevated G gamma globin content are tightly linked to this haplotype, we studied 55 members of five Saudi families in which sickle cell disease is present. The results did not suggest a tight linkage of the haplotype to increased fetal hemoglobin production. On the other hand, several sickle trait family members heterozygous for the haplotype had normal fetal hemoglobin production in culture but elevated G gamma to A gamma ratios in peripheral blood. This observation suggests that in this genetic background increased expression of the G gamma globin gene may occur without a measurable increase in total fetal hemoglobin production. The family studies also clearly demonstrate that increased fetal hemoglobin production by erythroid progenitors is dependent on zygosity for the sickle gene in this population. These findings strongly suggest that other factors, such as the products of genes stimulated by hemolytic stress or other genetic determinants associated with the Saudi beta S chromosome, may interact with the -158 C----T substitution and influence gamma globin gene expression in this population.

scholarly journals A potential regulatory region for the expression of fetal hemoglobin in sickle cell disease

Blood ◽  
1994 ◽  
Vol 84 (1) ◽  
pp. 331-338 ◽  
Author(s):  
S Pissard ◽  
Y Beuzard
Keyword(s):  
Sickle Cell Disease ◽  
Protein Binding ◽  
Sickle Cell ◽  
Globin Gene ◽  
Regulatory Region ◽  
Fetal Hemoglobin ◽  
Beta Globin ◽  
Cell Disease ◽  
Hemoglobin F ◽  
Binding Motifs

Abstract We describe a 0.5-kb region located 1.65 to 1.15 kb upstream of the G gamma fetal globin gene with three polymorphisms of erythroid and ubiquitous nuclear protein binding motifs (GATA, CRE, and a new protein binding site). These three polymorphisms result in high-affinity and low-affinity motifs for nuclear proteins, and are combined in four arrangements called pre-G gamma frameworks (pG gamma Fs). Each pG gamma F is linked with one of the major haplotypes of the beta-globin gene cluster observed in sickle cell disease (SCD) associated with different mean levels of hemoglobin F (Hb F) expression (P < .001). This strong linkage and the differing affinities suggest that this region may be involved in the modulation of Hb F expression in SCD.

New Concepts in Genome Regulation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. SCI-47-SCI-47
Author(s):  
Ann Dean ◽  
Jongjoo Lee ◽  
Ryan Dale ◽  
Ivan Krivega
Keyword(s):  
Sickle Cell Disease ◽  
Long Range ◽  
Control Region ◽  
Sickle Cell ◽  
Globin Gene ◽  
Locus Control Region ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Cell Disease ◽  
Gamma Globin

Abstract Manipulating gene regulation to favor gamma-globin transcription over beta-globin transcription has been a goal of research in erythropoiesis for decades because of its relevance to amelioration of the pathophysiology of sickle cell disease and beta-thalassemia. A fundamental unanswered question in biology is how the unique pattern of gene expression, the transcriptome, of the many different individual mammalian cell types arises from the same genome blueprint and changes during development and differentiation. There is a growing appreciation that genome organization and the folding of chromosomes is a key determinant of gene transcription. Within this framework, enhancers function to increase the transcription of target genes over long linear distances. To accomplish this, enhancers engage in close physical contact with target promoters through chromosome folding, or looping. These long range interactions are orchestrated by cell type specific proteins and protein complexes that bind to enhancers and promoters and stabilize their interaction with each other. We have been studying LDB1, a member of an erythroid protein complex containing GATA1, TAL1 and LMO2. The LDB1complex activates erythroid genes through occupancy of virtually all erythroid enhancers. LDB1engages in homo- and heterotypic interactions with proteins occupying the promoters of erythroid genes to bring them into proximity with their enhancers. We find that enhancer long range looping activity can be redirected. Both targeting of the beta-globin locus control region to the gamma-globin gene in adult erythroid cells by the tethering of LDB1 or epigenetic unmasking of a silenced gamma-globin gene lead to increased locus control region (LCR)/gamma-globin contact frequency and reduced LCR/beta-globin contact. The outcome of these manipulations is robust, pan-cellular gamma-globin transcription activation with a concomitant reduction in beta-globin transcription. These examples suggest that chromosome looping can be considered a therapeutic target for gene activation or gene silencing to ameliorate genetic diseases such as sickle cell disease and beta-thalassemia. Disclosures No relevant conflicts of interest to declare.

scholarly journals Fetal hemoglobin levels and beta s globin haplotypes in an Indian populations with sickle cell disease

Blood ◽  
1987 ◽  
Vol 69 (6) ◽  
pp. 1742-1746
Author(s):  
AE Kulozik ◽  
BC Kar ◽  
RK Satapathy ◽  
BE Serjeant ◽  
GR Serjeant ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Sickle Cell Trait ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Globin Genes ◽  
Cell Disease ◽  
Hemoglobin F ◽  
S Haplotype ◽  
Asian Populations

To further explore the cause for variation in hemoglobin F (Hb F) levels in sickle cell disease, the beta globin restriction-fragment length polymorphism haplotypes were determined in a total of 303 (126 SS, 141 AS, 17 S beta degrees, 7 A beta, degrees and 12 AA) Indians from the state of Orissa. The beta s globin gene was found to be linked almost exclusively to a beta S haplotype ( -++-), which is also common in Saudi Arabian patients from the Eastern Province (referred to as the Asian beta s haplotype). By contrast, the majority of beta A and beta degree thalassemia globin genes are linked to haplotypes common in all European and Asian populations (+-----[+/-]; --++-++). Family studies showed that there is a genetic factor elevating Hb F levels dominantly in homozygotes (SS). This factor appears to be related to the Asian beta s globin haplotype, and a mechanism for its action is discussed. There is also a high prevalence of an independent Swiss type hereditary persistence of fetal hemoglobin (HPFH) determinant active in both the sickle cell trait and in sickle cell disease.

Effects of 5-aza-2′-deoxycytidine on fetal hemoglobin levels, red cell adhesion, and hematopoietic differentiation in patients with sickle cell disease

Blood ◽  
2003 ◽  
Vol 102 (12) ◽  
pp. 3865-3870 ◽  
Author(s):  
Yogen Saunthararajah ◽  
Cheryl A. Hillery ◽  
Don Lavelle ◽  
Robert Molokie ◽  
Louise Dorn ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Mechanism Of Action ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Red Cell ◽  
Cell Disease ◽  
Hemoglobin F ◽  
Total Hemoglobin

Abstract Fetal hemoglobin (HbF) decreases polymerization of sickle hemoglobin (HbS) and improves outcomes in sickle cell disease (SSD). Therefore, a therapeutic goal in SSD is pharmacologic reactivation of HbF. Silencing of the γ-globin (HbF) gene is associated with DNA methylation. The cytosine analog 5-aza-2′-deoxycytidine (decitabine) hypomethylates DNA by inhibiting DNA methyltransferase. We examined if subcutaneous decitabine could increase HbF levels and improve SSD pathophysiology without cytotoxicity. Eight symptomatic SSD patients resistant or intolerant of standard treatment with hydroxyurea received decitabine 0.2 mg/kg subcutaneously 1 to 3 times per week in 2 cycles of 6-week duration. Treatment decreased neutrophils and increased mean HbF (6.5% to 20.4%, P &lt; .0001) and mean total hemoglobin (76 to 96 g/L [7.6 to 9.6 g/dL], P &lt; .001). Features of vaso-occlusive crisis pathophysiology such as red cell adhesion, endothelial damage, and coagulation pathway activity significantly improved. γ-Globin gene promoter methylation decreased, and platelets and the proportion of megakaryocytes and erythroid cells in the marrow increased without a decrease in marrow cellularity, consistent with a DNA hypomethylating, noncytotoxic mechanism of action. Weekly subcutaneous decitabine produces cumulative increases in HbF and total hemoglobin through a noncytotoxic mechanism of action. Chronic dosing and sustained increases in hemoglobin F and total hemoglobin levels may be possible. Further studies in SSD and thalassemia are indicated.

scholarly journals Fetal hemoglobin levels and beta s globin haplotypes in an Indian populations with sickle cell disease

Blood ◽  
1987 ◽  
Vol 69 (6) ◽  
pp. 1742-1746 ◽  
Author(s):  
AE Kulozik ◽  
BC Kar ◽  
RK Satapathy ◽  
BE Serjeant ◽  
GR Serjeant ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Sickle Cell Trait ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Globin Genes ◽  
Cell Disease ◽  
Hemoglobin F ◽  
S Haplotype ◽  
Asian Populations

Abstract To further explore the cause for variation in hemoglobin F (Hb F) levels in sickle cell disease, the beta globin restriction-fragment length polymorphism haplotypes were determined in a total of 303 (126 SS, 141 AS, 17 S beta degrees, 7 A beta, degrees and 12 AA) Indians from the state of Orissa. The beta s globin gene was found to be linked almost exclusively to a beta S haplotype ( -++-), which is also common in Saudi Arabian patients from the Eastern Province (referred to as the Asian beta s haplotype). By contrast, the majority of beta A and beta degree thalassemia globin genes are linked to haplotypes common in all European and Asian populations (+-----[+/-]; --++-++). Family studies showed that there is a genetic factor elevating Hb F levels dominantly in homozygotes (SS). This factor appears to be related to the Asian beta s globin haplotype, and a mechanism for its action is discussed. There is also a high prevalence of an independent Swiss type hereditary persistence of fetal hemoglobin (HPFH) determinant active in both the sickle cell trait and in sickle cell disease.

scholarly journals Genome editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: An approach for treating sickle cell disease and β-thalassemia

2016 ◽  
Vol 113 (38) ◽  
pp. 10661-10665 ◽  
Author(s):  
Lin Ye ◽  
Jiaming Wang ◽  
Yuting Tan ◽  
Ashley I. Beyer ◽  
Fei Xie ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Genome Editing ◽  
Sickle Cell ◽  
Globin Gene ◽  
Autologous Transplantation ◽  
Clinical Manifestations ◽  
Fetal Hemoglobin ◽  
Compound Heterozygous ◽  
Cell Disease ◽  
Hematopoietic Stem

Hereditary persistence of fetal hemoglobin (HPFH) is a condition in some individuals who have a high level of fetal hemoglobin throughout life. Individuals with compound heterozygous β-thalassemia or sickle cell disease (SCD) and HPFH have milder clinical manifestations. Using RNA-guided clustered regularly interspaced short palindromic repeats-associated Cas9 (CRISPR-Cas9) genome-editing technology, we deleted, in normal hematopoietic stem and progenitor cells (HSPCs), 13 kb of the β-globin locus to mimic the naturally occurring Sicilian HPFH mutation. The efficiency of targeting deletion reached 31% in cells with the delivery of both upstream and downstream breakpoint guide RNA (gRNA)-guided Staphylococcus aureus Cas9 nuclease (SaCas9). The erythroid colonies differentiated from HSPCs with HPFH deletion showed significantly higher γ-globin gene expression compared with the colonies without deletion. By T7 endonuclease 1 assay, we did not detect any off-target effects in the colonies with deletion. We propose that this strategy of using nonhomologous end joining (NHEJ) to modify the genome may provide an efficient approach toward the development of a safe autologous transplantation for patients with homozygous β-thalassemia and SCD.

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