Hereditary Persistence Of Fetal Hemoglobin Research Articles

Clinical variability and molecular characterization of Hbs/Gγ (Aγδβ)0-thal and Hbs/HPFH in Indian sickle cell disease patients: AIIMS experience

ABSTRACTIntroduction: In sickle cell disease (SCD) patients, among the predictors of survival, HbF levels play a significant role in lowering the morbidity and mortality. Coinheritance of δβ thalassemia and hereditary persistence of fetal hemoglobin (HPFH) may contribute to variable HbF levels in SCD patients, thus influencing their clinicopathological profile. Such cases are sparsely documented in the literature and thus, we screened the presence of δβ thalassemia and HPFH in 126 cases of SCD with high HbF.Material and methods: A total 126 SCD individuals with raised HbF levels were the study subject. Capillary zone electrophoresis (CZE) was done for the quantitative assessment of hemoglobin variants. HbSC, HbSD, HbAS and HbSE cases were excluded. Asian Indian Gγ(Aγδβ)0-thal, δβ0-thal (Sicilian, 13.4 kb), (Chinese, 100 kb), HPFH-1 (Black, 106 kb), HPFH-2 (Ghanaian, 105 kb), HPFH-3 (Indian, 48.5 kb) were done by GAP-PCR.Results: Out of 126, 78 cases (62%) were homozygous for SCD. The remaining 48 cases suspected to be heterozygous were furthered screened and 6/48 cases (12.5%) were found to be compound heterozygous. Out of these 6 cases,4(66.66%) had HbS/ δβ- Gγ(Aγδβ)0 and 2(33%) had HbS/HPFH compound heterozygous condition. None of the patients had δβ0-thal (Sicilian, 13.4 kb), (Chinese, 100 kb), HPFH-1 (Black, 106 kb), HPFH-2 (Ghanaian, 105 kb).Conclusion: This study highlights the importance of understanding the complex patho-physiology of compound heterozygous cases of HbS/HPFH and HbS/δβ thalassemia, as these infrequent conditions lead to change in phenotype and clinical severity of the disease. Insight into more such cases will open the window to better analyze the disease pathogenesis in these rare compound heterozygous conditions, as this will be beneficial to formulate proper management protocol in these patients.

Co-inheritance of Hereditary Persistence of Fetal Hemoglobin and Beta Thalassemia: A Rare Association

Co-inheritance of Hereditary Persistence of Fetal Hemoglobin and Beta Thalassemia: A Rare Association

TALEN-Mediated Gene Editing of HBG in Human Hematopoietic Stem Cells Leads to Therapeutic Fetal Hemoglobin Induction

Elements within the γ-hemoglobin promoters (HBG1 and HBG2) function to bind transcription complexes that mediate repression of fetal hemoglobin expression. Sickle cell disease (SCD) subjects with a 13-bp deletion in the HBG1 promoter exhibit a clinically favorable hereditary persistence of fetal hemoglobin (HPFH) phenotype. We developed TALENs targeting the homologous HBG promoters to de-repress fetal hemoglobin. Transfection of human CD34+ cells with TALEN mRNA resulted in indel generation in HBG1 (43%) and HBG2 (74%) including the 13-bp HPFH deletion (∼6%). Erythroid differentiation of edited cells revealed a 4.6-fold increase in γ-hemoglobin expression as detected by HPLC. Assessment of TALEN-edited CD34+ cells in vivo in a humanized mouse model demonstrated sustained presence of indels in hematopoietic cells up to 24 weeks. Indel rates remained unchanged following secondary transplantation consistent with editing of long-term repopulating stem cells (LT-HSCs). Human γ-hemoglobin expressing F cells were detected by flow cytometry approximately 50% more frequently in edited animals compared to mock. Together, these findings demonstrate that TALEN-mediated indel generation in the γ-hemoglobin promoter leads to high levels of fetal hemoglobin expression in vitro and in vivo, suggesting that this approach can provide therapeutic benefit in patients with SCD or β-thalassemia.

Persistence of CRISPR/Cas9-Edited Hematopoietic Stem and Progenitor Cells and Reactivation of Fetal Hemoglobin in Nonhuman Primates

Beta-thalassemia and sickle cell disease are monogenic disorders that are currently treated by allogeneic bone marrow (BM) transplantation although the challenges of finding a suitable matched-donor and the risk of graft vs host disease have limited the adoption of this otherwise curative treatment. A potentially promising approach for hemoglobinopathies aims to reactivate fetal hemoglobin (HbF) as a substitute for defective or absent adult hemoglobin by modifying the patient's own hematopoietic stem and progenitor cells (HSPCs). Here, we evaluated CRISPR/Cas9-induced small deletions in HSPCs that are associated with hereditary persistence of fetal hemoglobin (HPFH) using our nonhuman primate (NHP) stem cell transplantation and gene therapy model.The CRISPR/Cas9 nuclease platform was employed to recapitulate a natural genetic alteration identified in individuals with HPFH, consisting of a 13-nucleotide (nt) deletion in the gamma globin gene promoter. A first cohort of three rhesus macaques received 70-75% HPFH-edited BM-derived CD34+ HSPCs. All animals showed rapid hematopoietic recovery and peripheral blood (PB) editing levels stabilized at 12-30% for at least a year post transplantation (Figure 1). HbF production, determined by circulating F-cells, persisted at frequencies of 8-22% and correlated with in vivo PB editing. Robust engraftment of gene-edited HSPCs in the BM compartment was observed in all animals, with no measurable off-target activity or clonal expansion.We have recently shown, that the CD34+CD90+CD45RA- phenotype is exclusively required for short- and long-term multilineage reconstitution, significantly reduces the target cell number for gene therapy/editing and is conserved between human and NHP hematopoietic cells (Radtke et al., STM, 2017). To explore this cell population further, we transplanted a second cohort of three animals by sort-purifying and solely editing this hematopoietic stem cell (HSC)-enriched CD34+CD90+CD45RA- phenotype, thus reducing the number of target cells by over 10-fold without impacting hematopoietic recovery, engraftment, or HbF reactivation. In vivo levels of gene-edited PB started at less than 5% because of the high number of co-infused unmodified progenitor cells, but rapidly increased to about 50% within 1 week (Figure 1) and stabilized at levels comparable to the CD34 cohort. This data supports our interpretation that CD34+CD90+CD45RA- cells are the main cell population relevant for long-term reconstitution and an excellent target for improved and efficient gene therapy/editing.These results demonstrate robust engraftment and persistence of CD34+ HPSCs as well as HSC-enriched CD34+CD90+CD45RA- cells that have been CRISPR/Cas9-edited at the 13nt-HPFH site, with marked and stable HbF reactivation and no overt adverse effects in a NHP transplantation and gene therapy model. Most importantly, we validated our refined CD90+ target which reduces the need for editing reagents by 90% without compromising the gene modification and engraftment efficiencies. These are the first data in a clinically relevant large animal model to demonstrate the feasibility and clinical applicability of CRISPR/Cas9-mediated fetal hemoglobin reactivation. The successful targeting and engraftment of our HSC-enriched population should also have significant implications for gene therapy and editing of other genetic diseases.Figure 1:Tracking of HPFH editing in transplanted animals. A) Editing efficiency was longitudinally determined by next generation sequencing of the targeted locus in PB white blood cells from 2 cohorts of transplanted rhesus animals. Frequency is represented as the proportion of all sequence reads containing an edited locus. B) Normalized frequency of the desired 13nt-HPFH deletion in the same animals as shown in A). DisclosuresNegre:Bluebird Bio: Employment, Equity Ownership, Other: Salary. Adair:RX Partners: Honoraria; Miltenyi Biotec: Honoraria; Rocket Pharmaceuticals: Patents & Royalties: PCT/US2017/037967 and PCT/US2018/029983. Scharenberg:Generation Bio: Equity Ownership; Casebia Therapeutics: Employment; Alpine Immune Sciences: Equity Ownership. Kiem:Rocket Pharmaceuticals: Consultancy; Magenta: Consultancy; Homology Medicine: Consultancy.

Red Blood Cell Adhesion Is Increased in the Young Adult Population with Sickle Cell Disease

Background:Sickle cell disease (SCD) is characterized by abnormal cellular adhesion to the endothelium, contributing to progressive vasculopathy and vaso-occlusion. The progression of the underlying pathophysiology in SCD with age is not well understood. We evaluated red blood cell (RBC) adhesion at clinical baseline to laminin (LN) in children and adults with HbSS, using the SCD Biochip.1 The SCD Biochip is a microfluidic device that recapitulates physiologic flow and allows quantitation of RBC adhesion to biological surfaces.1Methods: This prospective cross-sectional study was conducted at The Children's Hospital at Montefiore in the Bronx, NY and University Hospitals Adult Sickle Cell Clinic in Cleveland, OH between 2014 and 2017. Blood samples were obtained from 29 children 8 to 18 years of age (33 samples, 28 HbSS and 1 HbSS HPFH (hereditary persistence of fetal hemoglobin), 13 males and 16 females), from 61 young adult patients 18 to 40 years of age (117 samples, 53 HbSS and 8 HbSS HPFH, 32 males and 29 females), and from 20 older adult patients >40 years of age (38 samples, 16 HbSS and 4 HbSS HPFH, 9 males and 11 females). All blood samples were obtained at clinical baseline. Of the children, young adult, and older adult populations, 45%, 46%, and 40% were on hydroxyurea treatment, respectively. Adhesion experiments were performed using surplus whole blood passed at physiological flow through LN-immobilized microchannels, and quantified after a wash step via microscope based on published protocols.1 Median values were used for multiple samples from a single individual.Results:Adults had higher, more heterogeneous RBC adhesion (440 ± 654, N=81) than did children (90 ± 193, N=29, not shown, p<0.001). Young adults tended to have higher adhesion than older adults (n.s.), as well as children (P<0.001, Figure 1). Young adults also had higher pain levels (relative to children, P=0.002) and higher reticulocyte counts (relative to older adults, Table 1, p=0.011), despite a higher total Hgb (P=0.012). As expected, children had a higher hemoglobin F level than young adults (Table 1, 11.1 ± 6.74 vs 4.85 ± 6.90, p=0.011).Conclusions:Our data demonstrates that adult patients with SCD have higher and more variable adhesion compared to pediatric patients with SCD, and this may be especially true in young adults. Older adults tended to have lower adhesion (perhaps due to compensatory genetic mutations that allowed them to survive before optimal pediatric care), but this was not statistically significant. Recall, as recently as the 1970s half of all Americans with SCD died before the age of 15 years of age. However, modern children with SCD are being treated aggressively with transfusions or hydroxyurea, and their low overall RBC adhesion reflects either these interventions or an innate low RBC adhesion during childhood. Increased adhesion in RBCs from young adults with SCD is congruent with increased mortality in the transition population2, and strongly suggests that modern treatments, as currently prescribed and taken, are insufficient to completely reverse the abnormal red cell physiology seen in young adults. Young adults have an increased RBC adhesion, possibly reflective of the natural history of SCD, and may benefit the most from anti-adhesive therapies and intensive interventions. Lower adhesion in children with SCD may also reflect an overall improved response to therapeutic interventions in children.

A Single -195 C < G HBG1 Promoter Mediated By CRISPR/Cas9 Genome Editing Induces Fetal Hemoglobin Synthesis in Hudep-2

Sickle cell disease (SCD) is a serious condition, chronic and undoubtedly represents a public health problem worldwide. SCD is caused by a point mutation in codon 6 of the β globin gene resulting in the production of a structurally abnormal hemoglobin, hemoglobin S. Although the cause of the disease has been known for more than fifty years, therapeutic options are still quite limited.High levels of fetal hemoglobin (HbF) in the blood are associated with a better clinical outcome in SCD patients. In some individuals, the expression of γ-globin gene persists into adulthood in elevated levels, which is called hereditary persistence of fetal hemoglobin (HPFH). A single nucleotide mutation from C to G at position -195 of the HBG1 gene promoter, called non deletional HPFH Brazilian type (nd-HPFH-B), augments the levels of HbF in patients in 7%- 30%. Nd-HPFH-B has been described by our group, but the mechanism and how this single mutation rises HbF levels differently in red blood cells is still unknown. Genome editing using CRISPR/Cas9 in HUDEP-2 cell, an erythroid precursor line, has been developed through homologous direct repair from a small single DNA strand containing the guanine in -195 position at HBG1 gene promotor. All the other genes, including the second HBG1 allele were unaltered.This point mutation has been carried out by CRISPR/Cas9 high fidelity system, capable of performing a specific break in the DNA target sequence, that improves homologous recombination rate of the donor sequence containing the -195 C<G mutation (ssODN -195).For the first time, we generated a HUDEP-2 cell line with the -195 C>T mutation in HBG1 promoter using CRISPR/Cas9 genome editing. The HUDEP-2 cells were nucleofected with Cas9 high fidelity ribonucleoprotein (104 pmol), crRNA:tracrRNA (120 pmol) complex and 1uM ssODN -195, using CD34+ human cell kit and program E-001 in AMAXA Nucleofector 4D- device (Lonza). Seven days after nucleofection, the transformed cells were submitted to clonal selection for 25 days. The genomic DNA from 48 clones were submitted by Sanger Sequencing. The sequencing analysis showed highest Crispr/Cas9 efficiency in genomic DNA cut (77.08%; 37/48) and satisfactory ssODN -195 homologous recombination (10.4%; 5/48).Five nd-HPFH-B HUDEP-2 clones and three other clones without the mutation, but with indels after Cas9 DNA cut (controls), were expanded in culture and the HbF levels were measure with anti-HbF antibody by flow cytometry in two biological replicates. HbF levels in nd-HPFH-B HUDEP-2 clones were 6.02%±1.4, 8.25% ± 0.28, 10.18% ± 3.71, 11.95% ± 0.49, 26,3% ± 4,6 while in controls were 1.69% ± 0.26, 1.66% ± 0.26, 0.59% ± 0.06. Two nd-HPFH-B clones were differentiated into erythrocyte in vitro, and fetal hemoglobin levels persisted at high levels seen previously.In addition, α-globin, β-globin and γ-globin mRNA levels were evaluated in three nd-HPFH-B HUDEP-2 clones and two control clones. The mRNA HBG1/HBG1+HBB percentage in nd-HPFH-B were 96.16% ± 4.10 against 22.63% ± 9.64 in controls.The monoallelic single nucleotide mutation -195 C>G is capable to increase the fetal hemoglobin levels up to 30% in nd-HPFH-B HUDEP-2, and our results shows that this is a potential experimental in vitro model to be used in future studies. DisclosuresNo relevant conflicts of interest to declare.

Comparative Studies Reveal Robust HbF Induction By Editing of HBG1/2 Promoters or BCL11A Erythroid-Enhancer in Human CD34+ Cells but That BCL11A Erythroid-Enhancer Editing Is Associated with Selective Reduction in Erythroid Lineage Reconstitution in a Xenotransplantation Model

▪Beta hemoglobinopathies resulting from dysfunctional or deficient adult beta-globin expression are some of the most prevalent inherited blood disorders in the world. Upregulation of developmentally-silenced fetal gamma-globin would replace adult beta-globin to ameliorate disease symptoms. One of the approaches to reactivate fetal globin expression in erythroid cells is through gene editing by zinc finger or CRISPR-Cas9 nucleases to disrupt the expression of a transcription factor BCL11A, which mediates fetal globin silencing. As BCL11A-deficiency leads to hematopoietic stem cells (HSCs) defects, the current editing approaches target the BCL11A erythroid-enhancer region located in intron 2 of the BCL11A gene to selectively reduce BCL11A expression in erythroid cells.Instead of targeting BCL11A, we sought to identify novel cis-regulatory elements at the beta-globin locus for targeted gene editing to achieve fetal globin reactivation. From a lenti-CRISPR mediated saturated mutagenesis screen covering the beta-globin locus using Human Umbilical Cord Blood-Derived Erythroid Progenitor (HUDEP)-2 cells, multiple fetal hemoglobin (HbF)-inducing genomic domains were identified. Most of the hits were concentrated at the gamma-globin (HBG1/2) promoters, clustered at known hereditary persistence of fetal hemoglobin (HPFH) mutation hotspots. In-depth genotype to phenotype analysis further defined the indels responsible for HbF induction in these subdomains. We interrogated multiple families of nucleases and guide RNA (gRNA) combinations with or without single-stranded oligodeoxynucleotides (ssODN) to guide editing outcome. gRNAs were selected based on their HbF induction potential (up to 40%) when introduced into mobilized peripheral blood (mPB) CD34+ hematopoietic stem and progenitor cells (HSPCs) as ribonucleoprotein (RNP) complexes. HSPCs transfected with RNPs targeting either the BCL11A erythroid-enhancer or the HBG1/2 proximal regions were then injected into NBSGW mice to study the editing in SCID-repopulating cells (SRC) and their multilineage reconstitution potential.All groups achieved high levels of human chimerism (>70% hCD45+/hCD45+mCD45) and comparable monocytes, granulocytes, B lymphocytes, and hCD34+ HSPCs reconstitution. However, BCL11A-edited cells showed selective reduction in erythroid lineage (CD235a+) output, up to 4-fold lower than untreated or HBG1/2 promoter-edited HSPCs. Sequencing analysis from lineage-specific sorted cells further revealed reduced editing levels at BCL11A erythroid-specific enhancer in the erythroid compartment compared to unfractionated bone marrow (BM) or other human lineages (70% in erythroid vs. 90% in all other lineages). Furthermore, the nonproductive fraction of indels that did not disrupt the BCL11A GATAA motif was significantly enriched in erythroid cells (22% in erythroid vs. 8% in other lineages). Ex vivo erythroid cultures suggests BCL11A erythroid-enhancer editing may lead to slightly increased apoptosis during erythroid differentiation. In contrast, HBG1/2 promoter-edited cells had similar editing levels and indel patterns across all lineages with no significant lineage skewing. When chimeric BM from HBG1/2 promoter-edited groups were cultured in erythroid conditions, ex vivo-derived erythroid cells had significantly elevated levels of HbF compared to controls. When CD235a+ cells were sorted without further culture from chimeric BM of mice engrafted with HBG1/2 promoter-edited cells, significantly increased levels of HbF were detected by UPLC (up to 30%) compared to controls (~6%). Thus, long-term HSCs have been edited productively at the HBG1/2 promoters. These cells were able to generate erythroid progenitors that harbor HbF-inducing indels, which in turn, gave rise to erythroid cells in vivo with a clinically-relevant levels of HbF in a xenotransplantation model.Together, our data suggest that BCL11A-edited cells have an erythroid differentiation defect or survival disadvantage in NBSGW mouse model that warrants further investigation. In contrast, editing of the HBG1/2 promoters in mPB CD34+ cells achieved sustained HbF expression in erythroid lineage while maintaining multilineage differentiation potential. Targeting of the HBG1/2 promoters in HSPCs may be an attractive strategy for the development of potential gene editing medicines for beta hemoglobinopathies. DisclosuresChang:Editas Medicine Inc.: Employment, Equity Ownership. Sanchez:Editas Medicine Inc.: Employment, Equity Ownership. Heath:Editas Medicine Inc.: Employment, Equity Ownership. deDreuzy:Editas Medicine Inc.: Employment, Equity Ownership. Haskett:Editas Medicine Inc.: Employment, Equity Ownership. Vogelaar:Editas Medicine Inc.: Employment. Gogi:Editas Medicine Inc.: Employment, Equity Ownership. Da Silva:Editas Medicine Inc.: Employment, Equity Ownership. Wang:Editas Medicine Inc.: Employment, Equity Ownership. Sadowski:Editas Medicine Inc.: Employment, Equity Ownership. Gotta:Editas Medicine Inc.: Employment, Equity Ownership. Siwak:Editas Medicine Inc.: Employment, Equity Ownership. Viswanathan:Editas Medicine Inc.: Employment, Equity Ownership. Loveluck:Editas Medicine Inc.: Employment, Equity Ownership. Chao:Editas Medicine Inc.: Employment, Equity Ownership. Tillotson:Editas Medicine Inc.: Employment, Equity Ownership. Chalishazar:Editas Medicine Inc.: Employment, Equity Ownership. Dass:Editas Medicine Inc.: Employment, Equity Ownership. Ta:Editas Medicine Inc.: Employment, Equity Ownership. Brennan:Editas Medicine Inc.: Employment, Equity Ownership. Tabbaa:Editas Medicine Inc.: Employment, Equity Ownership. Marco:Editas Medicine Inc.: Employment, Equity Ownership. Zuris:Editas Medicine Inc.: Employment, Equity Ownership. Reyon:Editas Medicine Inc.: Employment, Equity Ownership. Isik:Editas Medicine Inc.: Employment, Equity Ownership. Friedland:Editas Medicine Inc.: Employment, Equity Ownership. Ta:Editas Medicine Inc.: Employment, Equity Ownership. Harbinski:Editas Medicine Inc.: Employment, Equity Ownership. Giannoukos:Editas Medicine Inc.: Employment, Equity Ownership. Teixeira:Editas Medicine Inc.: Employment, Equity Ownership. Wilson:Editas Medicine Inc.: Employment, Equity Ownership. Albright:Editas Medicine Inc.: Employment, Equity Ownership. Jiang:Editas Medicine Inc.: Employment, Equity Ownership.

Reduced Plasmodium Falciparum Growth in Erythrocytes Is Associated with Fetal Hemoglobin Expression

Introduction: The risk of clinical Plasmodium falciparum malaria is significantly low in the first few months of life. Much of this protection has been attributed to maternal antibodies transmitted to the infant in utero or via breast milk, however less severe disease has also been associated with fetal hemoglobin (HbF). Impaired P. falciparum growth in cord blood erythrocytes and reduced digestion of HbF protein by recombinant plasmepsin II, a hemoglobinase, has also been observed in vitro, while in vivo studies have demonstrated decreased P. falciparum growth in high HbF-containing human gamma (γ)-transgenic murine red cells. Though inefficiency of hemoglobinase digestion of HbF appears plausible given enhanced alpha (α)/γ relative to α/beta (β) dimer and tetramer stability, it has been difficult to assess such inefficiency in vitro given the dramatic effect of current protease inhibitors on parasite growth. Instead, more focus has drifted towards oxidative stress and poor cytoadherence as mechanisms underlying the protection conferred by HbF. To test the hypothesis that HbF affects malaria infection, we mapped P. falciparum growth in cord blood and heterozygote A/HPFH erythrocytes using precise DNA replication measurements throughout the parasite blood stage cell-cycle.Methods: Adult (AA), cord, and heterozygote Hereditary Persistence of Fetal Hemoglobin (A/HPFH) samples were collected within one week of each other and within 1 month of assay date. The P. falciparum 3D7 IG06 parasite line (a gift from Daniel Goldberg, Washington University School of Medicine, St. Louis) was used for growth assays. All growth assays were performed at 1% oxygen using synchronized schizonts obtained by magnet purification via the MACS system. Average number of nuclei per parasite was determined via flow cytometry using SYBR Mean Fluorescence Intensity (MFI) as previously described.Results: By 32 hours post invasion into cord blood, a definite decrease in P. falciparum DNA replication, a sensitive marker of parasite growth, was observed compared to growth in adult erythrocytes (Figure 1A). Compared to the complete growth defect observed in deoxygenated sickle cell trait (AS), the almost 40% reduction (p=0.0002) in the average number of nuclei in cord blood RBCs was modest, allowing for growth through schizogony. The growth inhibition in cord blood erythrocytes was not sensitive to changes in oxygen levels (data not shown). Ring/early trophozoite stage parasites were morphologically similar in both adult and cord blood erythrocytes (Figure 1B), with growth differences beginning to be apparent between 28 and 32 hours, coincident with the onset of hemoglobin digestion. To ensure that the growth defect observed was not due to cord blood erythrocyte factors unrelated to HbF, we examined heterozygous A/HPFH RBCs which are similar to adult AA erythrocytes, but with expression of HbF. Parasites in A/HPFH erythrocytes (with ~20-30% HbF), demonstrated a 25% growth reduction in the average number of nuclei at 36hpi, compared to those in adult AA RBCs (Figure 2A and 2B), indicating the growth inhibitory effect of HbF.Conclusion: Though less striking compared to the severe stalling observed in deoxygenated AS RBCs, growth of P. falciparum 3D7 IG06 in cord blood erythrocytes is clearly delayed compared to growth in adult AA erythrocytes. This growth defect is oxygen independent, evidence that a different mechanism of parasite growth inhibition is involved than that observed in AS RBCs. The consistent growth delay observed in heterozygote A/HPFH confirms that parasite growth deficiency also occurs at lower percentages of HbF than those found in cord blood presumably because α2/γ-β heterodimers are also resistant to digestion by P. falciparum hemoglobinases. Finally, these data suggest that treatment of sickle cell disease by increasing fetal hemoglobin might be safer in malaria zones than would be employment of direct anti-sickling agents. [Display omitted] DisclosuresHankins:Global Blood Therapeutics: Research Funding; NCQA: Consultancy; bluebird bio: Consultancy; Novartis: Research Funding.

Emerging Genetic Therapy for Sickle Cell Disease.

The genetic basis of sickle cell disease (SCD) was elucidated >60 years ago, yet current therapy does not rely on this knowledge. Recent advances raise prospects for improved, and perhaps curative, treatment. First, transcription factors, BCL11A and LRF/ZBTB7A, that mediate silencing of the β-like fetal (γ-) globin gene after birth have been identified and demonstrated to act at the γ-globin promoters, precisely at recognition sequences disrupted in rare individuals with hereditary persistence of fetal hemoglobin. Second, transformative advances in gene editing and progress in lentiviral gene therapy provide diverse opportunities for genetic strategies to cure SCD. Approaches include hematopoietic gene therapy by globin gene addition, gene editing to correct the SCD mutation, and genetic manipulations to enhance fetal hemoglobin production, a potent modifier of the clinical phenotype. Clinical trials may soon identify efficacious and safe genetic approaches to the ultimate goal of cure for SCD.

A Rapid, Affordable and Feasible Method for Detection of the HBG1: g.-225_-222delAGCA Polymorphism

Single point mutations or small deletions in the Aγ - and Gγ-globin gene promoter region are associated to the nondeletional hereditary persistence of fetal hemoglobin (HPFH). Currently, DNA sequencing is most common technique adopted for detection of hemoglobin (Hb) mutations. However, some can be rapidly detected because they either destroy or create a recognition site for a restriction enzyme. Here we show that the 4 bp deletion, HBG1: g.-225_-222delAGCA in the Aγ-globin gene promoter can be easily detected using the Tru1I (MseI) restriction enzyme that cuts only in the absence of this deletion. This approach utilizes ordinary instrumentations (thermocycler and agarose gel electrophoresis) available in any basic molecular genetics laboratory, providing a reliable and inexpensive method of genetic screening.

Induction of fetal hemoglobin synthesis by CRISPR/Cas9-mediated editing of the human β-globin locus

AbstractNaturally occurring, large deletions in the β-globin locus result in hereditary persistence of fetal hemoglobin, a condition that mitigates the clinical severity of sickle cell disease (SCD) and β-thalassemia. We designed a clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) strategy to disrupt a 13.6-kb genomic region encompassing the δ- and β-globin genes and a putative γ-δ intergenic fetal hemoglobin (HbF) silencer. Disruption of just the putative HbF silencer results in a mild increase in γ-globin expression, whereas deletion or inversion of a 13.6-kb region causes a robust reactivation of HbF synthesis in adult erythroblasts that is associated with epigenetic modifications and changes in chromatin contacts within the β-globin locus. In primary SCD patient-derived hematopoietic stem/progenitor cells, targeting the 13.6-kb region results in a high proportion of γ-globin expression in erythroblasts, increased HbF synthesis, and amelioration of the sickling cell phenotype. Overall, this study provides clues for a potential CRISPR/Cas9 genome editing approach to the therapy of β-hemoglobinopathies.

Contrasting co-inheritance of alpha and beta mutations in delta beta thalassemia and hereditary persistence of fetal hemoglobin: a study from India

ABSTRACT Background and objectives: Coinheritance of δβ thalassemia and HPFH with inherited factors is sparsely documented and may affect treatment modalities. So, we screened the presence of α deletion and β mutations in δβ thalassemia and HPFH disorders in 52 cases with high Hb F concentration. Material and methods: Fifty-two individuals with raised HbF levels were study subjects. CZE was done for quantitative assessment of hemoglobin variants. Asian Indian inversion deletion break point type A, B and HPFH-3 were done by GAP-PCR. Results: 18/52 cases of δβ Gγ (Aγδβ)0 thalassemia and 28/52 cases of HPFH-3 deletion were characterized. 6/52 patients with raised HbF levels were negative for δβ Gγ (Aγδβ)0 and HPFH-3 deletion. 9/18 (50%) were heterozygous for Gγ(Aγδβ)0 break point type A, 6/18 (33%) were heterozygous for break point type B and 3/18 (17%) were homozygous. Of the nine patients heterozygous for Gγ(Aγδβ)0 break point type A, three (33%) patients were double heterozygous with alpha 3.7 kb deletion and two (22%) patients showed compound heterozygosity with IVS 1–5(G–C) mutation. 4/9 (45%) patients were Gγ(Aγδβ)0 heterozygous. Discussion and conclusion: We found 5/18(27.β) δβ-thalassemia cases with co-inherited alpha 3.7 deletion and 3/18 (16β) cases with IVS 1–5(G–C) mutation. Patients showed features of thalassemia intermedia phenotype among which those with co-inherited IVS 1–5(G–C) mutation showed severe phenotype as compared to those with co-inherited alpha 3.7 deletion. So, we highlight importance of genotyping of patients with δβ thalassemia or HPFH and coinheritance with inherited factors which plays crucial role in clinicopathological profile and setting up prenatal diagnostic protocol.

Natural regulatory mutations elevate the fetal globin gene via disruption of BCL11A or ZBTB7A binding.

β-hemoglobinopathies such as sickle cell disease (SCD) and β-thalassemia result from mutations in the adult HBB (β-globin) gene. Reactivating the developmentally silenced fetal HBG1 and HBG2 (γ-globin) genes is a therapeutic goal for treating SCD and β-thalassemia 1 . Some forms of hereditary persistence of fetal hemoglobin (HPFH), a rare benign condition in which individuals express the γ-globin gene throughout adulthood, are caused by point mutations in the γ-globin gene promoter at regions residing ~115 and 200 bp upstream of the transcription start site. We found that the major fetal globingene repressors BCL11A and ZBTB7A (also known as LRF) directly bound to the sites at -115 and -200 bp, respectively. Furthermore, introduction of naturally occurring HPFH-associated mutations into erythroid cells by CRISPR-Cas9 disrupted repressor binding and raised γ-globin gene expression. These findings clarify how these HPFH-associated mutations operate and demonstrate that BCL11A and ZBTB7A are major direct repressors of the fetal globin gene.

Switching from fetal to adult hemoglobin.

The switch from fetal to adult hemoglobin relies on repression or silencing of the upstream γ-globin gene, but identification of the transcriptional repressors that bind to the sites at which a cluster of naturally occurring variants associated with HPFH (hereditary persistence of fetal hemoglobin) are found has been elusive. A new study provides mechanistic evidence for the direct binding of BCL11A and ZBTB7A, two previously identified γ-globin gene repressors.

The relationship between the variations in Gγ and Aγ promotors and the Hereditary Persistence of Fetal Hemoglobin (HPFH).

In the present study, sixty-two samples that have 1.5% and upper level of fetal hemoglobin (HbF), were examined to investigate the relationship between HbF level and non-deletional mutations in both Gγ (G gamma) globin (HBG2) and Aγ (A gamma) globin (HBG1) genes. Four variations were observed in the promotor of Gγ gene, which are -158C/T, -309A/G, -369C/G, and -567T/G. Also, four variations were observed in the 5'-UTR (untranslated regions) and promotor of Aγ gene, which are +25G/A, -369G/C, -499T/A, and -588G/A. One -222/-225 AGCA del homozygous and six variations as heterozygous in A gamma globin gene promotor region were also observed. The results of the current study suggested that there was a significant relationship between high HbF levels and two variations (-309A/T and -369C/G) in Gγ gene promotor. Additionally, a significant relationship between two variations (+25G/A and -499T/A) in Aγ gene promotor was also observed. Furthermore, the persons who carry these variations with high levels of HbF indicated that there might be a haplotype effect between these variations.

The Results of Hemoglobin Variant Analysis in Patients Revealing Microcytic Erythrocytosis on Complete Blood Count.

Microcytic erythrocytosis is an underrecognized and underevaluated complete blood count (CBC) finding. The literature pertaining to the determination of its etiology specifically by hemoglobin variant analysis is limited. We performed hemoglobin variant analysis by high performance liquid chromatography on 137 patients who revealed microcytic erythrocytosis on CBC, and reviewed the results for the diagnosis of hemoglobin-associated disorders. A diagnosis of thalassemia trait and/or a hemoglobinopathy was established in 93 of 137 (67.9%) patients. Amongst these, ß-thalassemia trait topped the list with 69 cases (74.1%), followed by hereditary persistence of fetal hemoglobin with 5 cases (5.5%), Hemoglobin E disease with 4 cases (4.3%), and ∂/ß-thalassemia with 2 cases (2.1%). Compound heterozygous conditions with 1 or more hemoglobinopathies and/or thalassemias were diagnosed in 13 cases (14.0%). Abnormal hemoglobins in the compound heterozygosity group included C, S, HPFH, and 2 unknowns. Hemoglobin variant analysis provided a very high positive yield in determining the etiology of microcytic erythrocytosis.

Prevention of Transcriptional γ-globin Gene Silencing by Inducing The Hereditary Persistence of Fetal Hemoglobin Point Mutation Using Chimeraplast-Mediated Gene Targeting.

Objective Hemoglobin F (HbF) augmentation is considered a clinically beneficial phenomenon in β-hemoglobinopathies. Prevention of γ-globin gene silencing, inspired by the hereditary persistence of fetal hemoglobin, may be a suitable strategy to upregulate HbF expression in these patients. Therefore, our objective was to assess the potential feasibility of induced -117 G→A substitution in HBG promoter in prevention of transcriptional silencing of the γ-globin. Materials and Methods In this experimental study, human peripheral blood-derived hematopoietic stem cells (HSCs) and the K562 cell line were differentiated to erythroid cells. Erythroid maturation was examined using cell morphology parameters and flow cytometry analysis of CD235a expression. A synthesised chimeraplast was transfected to differentiating cells. The efficiency of chimeraplast delivery into target cells was assessed by flow cytometry. Restriction-fragment length polymorphism and DNA sequencing verified oligonucleotide-directed mutagenesis. Gene conversion frequency and globin genes expression was quantified through Allele specific-quantitaive polymerase chain reaction (AS-qPCR) and quantitative-PCR respectively. Results Increase in CD235a-expressing cells along with observations made for different stages of erythroid maturation confirmed erythroid differentiation in HSCs and K562 cells. γ to β-globin gene switching was estimated to be on days 18-21 of HSC differentiation. Flow cytometry analysis showed that more than 70% of erythroid progenitor cells (EPCs) were transfected with the chimeraplast. The highest gene conversion efficiency was 7.2 and 11.1% in EPCs and K562 cells respectively. The induced mutation led to a 1.97-fold decrease in β/γ-globin gene expression in transfected EPCs at the experimental end point (day 28) whereas, due to the absence of β-globin gene expression following K562 differentiation, this rate was not evaluable. ConclusionOur results suggest the effectiveness of chimeraplasty in induction of the mutation of interest in both EPCs and K562 cells. We also demonstrate that the single nucleotide promoter variant was able to significantly inhibit γ-globin gene silencing during erythroid differentiation.

Low Impact of Genetic Modifiers on the Phenotype of Homozygous Beta Thalassemia in the Last Decennial Cohort of Thalassemia Newborns in Sardinia

Historically 15-20% of homozygous ß-thalassemia in Sardinia developed the phenotype of thalassemia intermedia not requiring transfusion for survival. However, this category of patients has broad and heterogeneous spectrum severity, embracing truly mild thalassemia patients and patients with severity similar to thalassemia major. Known genetic modifiers that ameliorate the severity of b-thalassemia are the mild genotypes of ß+-thalassemia, the coinheritance of α-thalassemia, of hereditary persistence of fetal hemoglobin (HPFH) and of polymorphic variants in the BCL11a, MYB and HBG1 genes. All together this modifiers allow the calculation of a genetic score of severity that predicts the possibility of remaining transfusion free at a given age, likely aiding in the clinical decision to start or delay the initiation of a stable transfusion program. In this study we planned to evaluate the ameliorating effects of these genetic modifiers on the thalassemia phenotype by prospectively evaluating 51 homozygous ß-thalassemia newborns followed at our Institution in the last ten years. All newborns were genotyped soon after birth for all known genetic modifiers and monitored clinically for the appearance of anemia or other complications with monthly check-ups from birth until they required transfusions, according to the TIF guidelines. In this decennial cohort, all but 2 patients younger than 20 months started a regular transfusion program before 36 months of age. Hence, the percentage of thalassemia intermedia in Sardinia has dropped in the last decade from 18 to 4% and might even reach 0% if the 2 patients non yet transfused will transfuse within the next year. This shift from thalassemia intermedia to major is mainly accounted for by the great improvement in mortality, morbility and quality of life for thalassemia major compared to thalassemia intermedia patients who frequently develop difficult to manage complications with advancing age. In the cohort, the reason for start of blood transfusions was anemia lower than 7gr/dl in 14 % of cases and the appearance of skeletal modifications or growth failure in the remaining 84% of patients. Thus, differently from the past, most patients were transfused despite an acceptable grade of anemia. As expected, the presence of a greater number of genetic modifiers significantly correlated with a delay in the time lag to the first transfusion. Even better positive correlation (p<0.0009) was found between the number of genetic modifiers and the levels of hemoglobin before beginning transfusion, confirming the positive influence of genetic modifiers on hemoglobin production. Since the start of transfusions, blood consumption did not positively correlated with the number of favorable genetic hits, but showed a trend toward significance, likely to achieve significance with increasing number of patients. Since the clinical decision to transfuse was taken mainly on the highly subjective parameter of appearance of skeletal modifications, it is possible that were transfused even the patients likely to have mild thalassemia intermedia and a good quality of life for most of their lifespan. We propose that at least the thalassemia patients with the best hemoglobin values (higher than 9 gr/dl) and a favorable background with at least 2 genetic modifiers (12% of our ߰-thalassemia newborns) should be more carefully evaluated for the possibility to evolve into a mild thalassemia intermedia phenotype with good quality of life for most of their lifespan. DisclosuresNo relevant conflicts of interest to declare.

Comparison of Fetal and Adult Erythroid Chromosomal Architectures Identifies a Novel Fetal Hemoglobin Regulatory Region

Chromatin structure is tightly intertwined with transcription regulation. The extent to which global chromatin architecture is subjected to alterations at different developmental stages within the same cell lineage has not been examined in great depth. Erythropoiesis offers an ideal model system to study the molecular mechanisms of gene regulation within the same cell lineage during development. Here, we comparatively defined via RNA-seq the transcriptomes, and via Hi-C and Capture-C the chromosome architectures of primary human fetal and adult erythroid cells.Overall, fetal and adult chromosomal conformations displayed a high degree of similarity. This includes the maintenance of A and B compartments representing active and inactive chromatin regions, respectively. Only ~5% of the genome switched compartments from A to B or vice versa, in agreement with the highly similar gene expression profiles. Moreover, topologically associating domains (TADs) were extensively preserved from fetal to adult stages. The developmentally regulated β-globin gene cluster is contained within one topologically associating domain (TAD) but folds into a three sub-TADs structure, the central one of which encompasses the β-globin locus. Notably, although the three sub-TAD structures are flanked by tissue invariant CTCF bound sites, they engage in looped contacts only in erythroid cells, indicating that erythroid specific transcription factors are required for CTCF mediated boundary contacts.At a finer scale, Capture-C detected distinct folding patterns at the developmentally controlled β-globin locus, including the expected stage-specific interactions between the enhancer (LCR) and the fetal γ-globin and adult β-globin genes. Importantly, we identified new developmental stage-specific chromatin contacts involving a region compassing a pseudogene (HBBP1) that resides between the fetal and adult globin genes. Specifically, HBBP1 engages in fetal stage-specific contacts with DNase hypersensitive sites HS5 and 3'HS1 while contacting the embryonic ε-globin gene at the adult stage. Deletion of a 2.3kb fragment encompassing HBBP1 (but not its transcriptional silencing) leads to strong reactivation of γ-globin gene expression in an adult erythroid cell line. This is accompanied by an architecturally restructured locus, including increased LCR-γ-globin chromatin interactions. Notably, the effects of HBBP1 deletion on chromatin architecture and gene expression closely mimic those of deleting the fetal globin repressor BCL11A, implicating BCL11A in the function of the HBBP1 region.In sum, our results identify a new segment, distinct from previously described regions linked to hereditary persistence of fetal hemoglobin, which engages in functionally important chromatin contacts. Since the HBBP1 region resides quite distantly from the structural globin genes, it might be a useful target for therapeutic genome editing without risking damage to the globin genes. Finally, our study highlights the power of high resolution chromosome architectural analysis to identify new regulatory regions. DisclosuresNo relevant conflicts of interest to declare.

KLF1 drives the expression of fetal hemoglobin in British HPFH

Abstractβ-Hemoglobinopathies are among the most common single-locus inherited diseases. In this condition, high fetal hemoglobin (HbF) levels have been found to be beneficial, and boosting HbF expression is seen as an attractive therapy. Naturally occurring mutations in the fetal globin promoter can result in high HbF persisting into adulthood in a benign condition known as hereditary persistence of fetal hemoglobin (HPFH). Individuals with one form of HPFH, British HPFH, carry a T to C substitution at position −198 of the fetal globin gene promoter. These individuals exhibit HbF levels of up to 20%, enough to ameliorate the symptoms of β-hemoglobinopathies. Here, we use clustered regularly interspaced short palindromic repeat–mediated genome editing to introduce the −198 substitution into human erythroid HUDEP-2 cells and show that this mutation is sufficient to substantially elevate expression of HbF. We also examined the molecular mechanism underlying the increase in fetal globin expression. Through a combination of in vitro and in vivo studies, we demonstrate that the mutation creates a de novo binding site for the important erythroid gene activator Krüppel-like factor 1 (KLF1/erythroid KLF). Our results indicate that introducing this single naturally occurring mutation leads to significantly boosted HbF levels.