Hereditary Persistence Of Fetal Hemoglobin Research Articles

Ftx-6058 Induces Fetal Hemoglobin Production and Ameliorates Disease Pathology in Sickle Cell Mice

Sickle cell disease (SCD) is a genetic disorder of the red blood cells caused by a mutation in the HBB gene, which results in red blood cell sickling, hemolysis, vaso-occlusive crises (VOCs), and other complications. Increasing HbF has the potential to prevent or reduce disease-related pathophysiology, including the risk of recurring events such as hemolysis and VOCs. Individuals with SCD who also have hereditary persistence of fetal hemoglobin (HPFH) and exhibit HbF levels of 25 - 30% are often asymptomatic from their SCD, underscoring the protective effect of increased HbF in SCD. Preclinical results with FTX-6058 demonstrate robust target engagement and corresponding increases in HbF levels up to approximately 40% of total hemoglobin, demonstrating the potential to have a significant impact in people living with SCD.FTX-6058 is an investigational, potent, and selective oral small-molecule inhibitor of Embryonic Ectoderm Development (EED) that has been demonstrated to induce robust fetal hemoglobin (HbF) protein expression in cell and murine models of SCD. Here, we report the in vivo effects of EED modulation, and subsequent polycomb repressive complex 2 (PRC2) inhibition in the Townes mouse model of Sickle Cell Disease. Compared with untreated and hydroxyurea treated animals, FTX-6058 (QD, 5mg/kg) exhibits potent target engagement as evidenced by ~70% reduction in H3K27me3 levels, the key epigenetic mark catalyzed by PRC2. Consistent with the robust target engagement observed with FTX-6058, a 2 - 3 fold increase in F cells and HbF protein were observed after 13 and 21 days of FTX-6058 treatment, which was pharmacologically superior to the fetal hemoglobin induction observed with 100 mg/kg hydroxyurea (~25% relative increase in F cells and HbF protein). A linear correlation was also observed between F cells and HbF protein production with FTX-6058 treatment, further supporting the robust HbF induction observed. Consistent with SCD disease presentation, Townes model mice have high reticulocyte counts as a result of premature RBC destruction and hemolysis. Townes model mice treated for 21 days with FTX-6058 ameliorated hemolysis as evidenced by decreases in reticulocytes and increases in red blood cells and total hemoglobin levels. Furthermore, FTX-6058 demonstrated the ability to impact inflammatory drivers of disease as evidenced by decreases in neutrophils and white blood cells. FTX-6058 did not have effects on other hematopoietic lineages. FTX-6058 also impacted pathophysiologic symptoms (e.g., splenomegaly) associated with SCD, as evidenced by ~25% reduction in spleen weight. While some trends were detected, no statistically significant changes were observed in hematological parameters or pathophysiologic symptoms with hydroxyurea, the current standard of care in SCD. These results observed with hydroxyurea underscore the need for new, novel therapies in SCD and other hemoglobinopathies.Taken together, these studies further support the therapeutic rationale of PRC2 modulation in SCD. The fetal hemoglobin induction and effects on hematological parameters and pathophysiologic symptoms observed with FTX-6058 have the potential to translate to meaningful clinical benefits in SCD and other hemoglobinopathies. DisclosuresMatson: Fulcrum Therapeutics, Inc.: Current Employment. Xie: Fulcrum Therapeutics, Inc.: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Roth: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Stuart: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Bruno: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Efremov: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Thompson: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Silver: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Moxham: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company.

Preclinical Development of EDIT301, an Autologous Cell Therapy Comprising AsCas12a-RNP Modified Mobilized Peripheral Blood-CD34 + Cells for the Potential Treatment of Transfusion Dependent Beta Thalassemia

Beta thalassemia is one of the most common recessive hematological disorders in the world with more than 200 mutations identified to date. These mutations reduce or completely abrogate beta globin expression. As beta globin pairs with alpha globin to form adult hemoglobin (HbA, α2β2), reduced or absent beta globin results in excessive alpha globin chains, which form toxic aggregates. These aggregates cause maturation blockade and premature death of erythroid precursors, and hemolysis of red blood cells (RBC), leading to varying degrees of anemia. Patients with the most severe form of beta thalassemia, namely beta thalassemia major, are transfusion-dependent, i.e., requiring life-long RBC transfusions accompanied by the burden of iron chelation therapy.EDIT-301 is an experimental autologous cell therapy in which CD34 + cells are genetically modified to promote gamma globin expression. EDIT-301 is currently in clinical development for sickle cell disease, and IND enabling stage for transfusion-dependent beta thalassemia (TDT). Gamma globin decreases the alpha to beta globin chain imbalance in beta thalassemia by pairing with the over-abundant alpha globin chains to form fetal hemoglobin (HbF, α2γ2). Gamma globin induction, and consequently HbF induction, for EDIT-301 is achieved through AsCas12a ribonucleoprotein (RNP)-mediated editing of the distal CCAAT box region of the HBG1 and HBG2 promoters, where naturally occurring hereditary persistence of fetal hemoglobin (HFPH) mutations exist. We chose this target over BCL11A based on previous preclinical data demonstrating that BCL11A editing reduces erythroid output in NBSGW mice. An engineered AsCas12a RNP edits the HBG1 and HBG2 promoter distal CCAAT box with high efficiency and specificity. We have previously shown that on-target editing of >80% was achieved in mobilized peripheral blood (mPB) CD34 + cells from normal donors with no detectable off-target editing both at research scale and at clinical manufacturing scale. Edited normal donor CD34 + cells led to long-term, polyclonal, multilineage engraftment without lineage skewing in immunocompromised mice and sustained robust HbF production in their erythroid progeny.To test whether EDIT-301 may be an efficacious therapy for TDT, mPB CD34 + cells from individuals with TDT were electroporated with the engineered AsCas12a RNP targeting the HBG1 and HBG2 promoters. AsCas12a RNP edited mPB CD34 + cells from individuals with TDT as efficiently as CD34 + cells from normal donors. Importantly, EDIT-301 has the potential to address the underlying pathophysiology of TDT, i.e., the maturation blockade and premature death of erythroid precursors. Erythroid differentiation of edited beta thalassemia CD34 + cells showed significant improvement in erythroid maturation and health. Specifically, ~70% edited erythroblasts reached late erythroblast stage compared to ~53% unedited erythroblasts; ~56% edited erythroid cells underwent terminal maturation and enucleated compared to ~28% of unedited erythroid cells; and non-viable erythroblasts decreased from ~33% to ~22% after editing. The improved erythropoiesis was accompanied by significantly increased total hemoglobin content per cell. These data strongly support that editing of the HBG1 and HBG2 promoter CCAAT box using engineered AsCas12a RNP can reverse the dyserythropoiesis associated with beta thalassemia and increase the hemoglobin production.In summary, we have provided strong preclinical data supporting the development of EDIT-301 for the treatment of TDT. Edited mPB CD34 + cells retained their ability to engraft without lineage skewing, resulted in robust HbF induction long-term, improved erythropoiesis, and increased hemoglobin content in TDT erythroid cells. These data support that a single administration of EDIT-301 may have the potential to safely and effectively reverse dyserythropoiesis and ameliorate anemia in individuals with TDT long-term. Clinical studies to demonstrate the safety and efficacy of EDIT-301 in the treatment of TDT are currently being planned. DisclosuresSousa: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Janoudi: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. deDreuzy: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Shearman: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zhang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Chang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

Primary myelofibrosis in untreated sickle cell disease: Are adult patients at higher risk for developing hematological myeloid neoplasms?

Primary myelofibrosis in untreated sickle cell disease: Are adult patients at higher risk for developing hematological myeloid neoplasms?

3'HS1 CTCF binding site in human β-globin locus regulates fetal hemoglobin expression.

Mutations in the adult β-globin gene can lead to a variety of hemoglobinopathies, including sickle cell disease and β-thalassemia. An increase in fetal hemoglobin expression throughout adulthood, a condition named hereditary persistence of fetal hemoglobin (HPFH), has been found to ameliorate hemoglobinopathies. Deletional HPFH occurs through the excision of a significant portion of the 3' end of the β-globin locus, including a CTCF binding site termed 3'HS1. Here, we show that the deletion of this CTCF site alone induces fetal hemoglobin expression in both adult CD34+ hematopoietic stem and progenitor cells and HUDEP-2 erythroid progenitor cells. This induction is driven by the ectopic access of a previously postulated distal enhancer located in the OR52A1 gene downstream of the locus, which can also be insulated by the inversion of the 3'HS1 CTCF site. This suggests that genetic editing of this binding site can have therapeutic implications to treat hemoglobinopathies.

Structural basis for human ZBTB7A action at the fetal globin promoter.

SUMMARYElevated levels of fetal globin protect against β-hemoglobinopathies, such as sickle cell disease and β-thalassemia. Two zinc-finger (ZF) repressors, BCL11A and ZBTB7A/LRF, bind directly to the fetal globin promoter elements positioned at −115 and −200, respectively. Here, we describe X-ray structures of the ZBTB7A DNA-binding domain, consisting of four adjacent ZFs, in complex with the −200 sequence element, which contains two copies of four consecutive C:G base pairs. ZF1 and ZF2 recognize the 5′ C:G quadruple, and ZF4 contacts the 3′ C:G quadruple. Natural non-coding DNA mutations associated with hereditary persistence of fetal hemoglobin (HPFH) impair ZBTB7A DNA binding, with the most severe disruptions resulting from mutations in the base pairs recognized by ZF1 and ZF2. Our results firmly establish ZBTB7A/LRF as a key molecular regulator of fetal globin expression and inform genome-editing strategies that inhibit repressor binding and boost fetal globin expression to treat hemoglobinopathies.

Activation of γ-globin gene expression by GATA1 and NF-Y in hereditary persistence of fetal hemoglobin

Hereditary persistence of fetal hemoglobin (HPFH) ameliorates β-hemoglobinopathies by inhibiting the developmental switch from γ-globin (HBG1/HBG2) to β-globin (HBB) gene expression. Some forms of HPFH are associated with γ-globin promoter variants that either disrupt binding motifs for transcriptional repressors or create new motifs for transcriptional activators. How these variants sustain γ-globin gene expression postnatally remains undefined. We mapped γ-globin promoter sequences functionally in erythroid cells harboring different HPFH variants. Those that disrupt a BCL11A repressor binding element induce γ-globin expression by facilitating the recruitment of transcription factors NF-Y to a nearby proximal CCAAT box and GATA1 to an upstream motif. The proximal CCAAT element becomes dispensable for HPFH variants that generate new binding motifs for activators NF-Y or KLF1, but GATA1 recruitment remains essential. Our findings define distinct mechanisms through which transcription factors and their cis-regulatory elements activate γ-globin expression in different forms of HPFH, some of which are being recreated by therapeutic genome editing.

CRISPR/Cas9 Genome Editing to Treat Sickle Cell Disease and B-Thalassemia: Re-Creating Genetic Variants to Upregulate Fetal Hemoglobin Appear Well-Tolerated, Effective and Durable.

Persistent expression of fetal hemoglobin (HbF) beyond the neonatal period is a rare, naturally-occurring condition, referred to as the Hereditary Persistence of Fetal Hemoglobin, which substantially ameliorates the pathology of Sickle Cell Disease (SCD) and β-thalassemia (β-thal). Extensive human genetic and epidemiologic studies have demonstrated that this condition is associated with one of several specific point mutations or deletions that lead to expression of γ-globin, resulting in upregulation of HbF. Our strategy is to use the CRISPR/Cas9 genome editing technology in human primary CD34+ hematopoietic stem and progenitor cells (HSPCs) to efficiently re-create specific genetic variants associated with elevated HbF and to evaluate their safety, effectiveness and durability as a potential therapeutic strategy to treat SCD and β-thal.We have optimized CRISPR/Cas9 editing of human primary HSPCs mobilized from healthy donors to routinely achieve editing efficiencies of greater than 80%. Improved viability with no reduction in editing efficiency was achieved with Cas9 delivered as a recombinant protein as compared to Cas9 delivered as mRNA. We have further optimized the process at clinical scale in our manufacturing facility to achieve similar viability and editing efficiency with a GMP-compatible process. The high rate of editing in human primary HSPCs mobilized from healthy donors was associated with robust HbF expression following erythroid differentiation. In patient samples, clinically relevant increases in γ-globin mRNA to 39% (as a ratio of γ/α) in one β-thal patient sample and to 38±5% (Mean±SD, as a ratio of γ/(γ+β)) in six SCD patient samples were observed. Similarly high rate of editing was observed in the CD34+CD38-CD90+CD45RA- long-term repopulating subset of human primary HSPCs, as in the overall HSPC population (88±6% vs 90±4% Mean±SD, n=4). We have demonstrated the long-term persistence of transplanted cells in a NOD-SCID-Gamma (NSG) xeno-transplant mouse model at 16 weeks (44±12% of cells in controls versus 44±14% and 41±13% for two target modifications, Mean±SD, n=44-47 for each), and that the proportion of the various hematopoietic lineages derived from the transplanted edited human primary HSPCs was not affected by the editing process. Finally, the high editing rate of 85-90% persisted in human bone marrow cells at 16 weeks, demonstrating durability of the edited cells. Because the NSG model does not appropriately replicate erythroid development, we have also demonstrated normal erythroid potential of the engrafted human CD34+ cells isolated from mouse bone marrow in ex vivo differentiation assays, and the editing level is maintained in the erythroid lineage. In-depth analysis confirmed no detectable off-target sequence disruption at over 5,000 candidate off-target sites that were identified bioinformatically through homology to the on-target site or experimentally through a double strand break identification method. In vivo safety and toxicology studies are ongoing.Taken together, these findings show that a CRISPR/Cas9-based therapy has the potential to upregulate HbF and treat diseases of b-globin. We have optimized editing conditions to achieve high efficiency and viability of editing in human primary HSPCs without detectable off-target sequence disruption, and in turn, demonstrate the durability of this approach in vivo. We believe that the results to date support the initiation of clinical studies for a CRISPR/Cas9 treatment for patients with SCD and β-thal. DisclosuresLin:CRISPR Therapeutics: Employment. Paik:CRISPR Therapeutics: Employment. Mishra:CRISPR Therapeutics: Employment. Burkhardt:CRISPR Therapeutics: Employment. Kernytsky:CRISPR Therapeutics: Employment. Pettiglio:CRISPR Therapeutics: Employment. Chen:CRISPR Therapeutics: Employment. Tomkinson:CRISPR Therapeutics: Employment. Woo:CRISPR Therapeutics: Employment. Cortes:CRISPR Therapeutics: Employment. Tan:CRISPR Therapeutics: Employment. Borland:CRISPR Therapeutics: Employment. Klein:CRISPR Therapeutics: Employment. Yen:Vertex: Employment. Mahajan:Vertex: Employment. Chan:Vertex: Employment. Eustace:Vertex: Employment. Porteus:CRISPRTx: Equity Ownership. Chakraborty:CRISPR Therapeutics: Employment. Cowan:CRISPR Therapeutics: Employment. Novak:CRISPR Therapeutics: Employment. Lundberg:CRISPR Therapeutics: Employment.

Time-of-flight mass spectrometry in the diagnosis of haemoglobin Kenya

Haemoglobin (Hb) variants are routinely detected by electrophoresis and high performance liquid chromatography (HPLC) in clinical laboratories. However, further molecular testing is often required to achieve a diagnosis. For rare variants molecular analysis may involve investigation of both the α- and β-globin gene clusters, as well as multiple techniques to detect point mutations and gene duplications or deletions. Time-of-flight (TOF) mass spectrometry is used as a secondary test in our laboratory to guide molecular analyses, saving both time and money. We present a case of Hb Kenya, a rare variant not typically seen in our patient population. Hb Kenya is associated with hereditary persistence of fetal haemoglobin (HPFH) and is the product of a γβ fusion gene arising from abnormal crossing over during meiosis. This variant is not easily diagnosed by routine haematology or genetic methods and may be confused with other disorders. TOF mass spectrometry was used to identify the variant mass, enabling appropriate molecular testing to be designed for confirmation of the diagnosis. This case highlights the utility of TOF mass spectrometry for haemoglobinopathy and thalassaemia investigations.

Enhanced HbF reactivation by multiplex mutagenesis of thalassemic CD34+ cells in vitro and in vivo

AbstractThalassemia or sickle cell patients with hereditary persistence of fetal hemoglobin (HbF) have an ameliorated clinical phenotype and, in some cases, can achieve transfusion independence. Inactivation via genome editing of γ-globin developmental suppressors, such as BCL11A or LRF/ZBTB7A, or of their binding sites, have been shown to significantly increase expression of endogenous HbF. To broaden the therapeutic window beyond a single-editing approach, we have explored combinations of cis- and trans-editing targets to enhance HbF reactivation. Multiplex mutagenesis in adult CD34+ cells was well tolerated and did not lead to any detectable defect in the cells' proliferation and differentiation, either in vitro or in vivo. The combination of 1 trans and 1 cis mutation resulted in high editing retention in vivo, coupled with almost pancellular HbF expression in NBSGW mice. The greater in vivo performance of this combination was also recapitulated using a novel helper-dependent adenoviral-CRISPR vector (HD-Ad-dualCRISPR) in CD34+ cells from β-thalassemia patients transplanted to NBSGW mice. A pronounced increase in HbF expression was observed in human red blood cells in mice with established predominant β0/β0-thalassemic hemopoiesis after in vivo injection of the HD-Ad-dualCRISPR vector. Collectively, our data suggest that the combination of cis and trans fetal globin reactivation mutations has the potential to significantly increase HbF both totally and on a per cell basis over single editing and could thus provide significant clinical benefit to patients with severe β-globin phenotype.

Molecular analysis of the erythroid phenotype of a patient with BCL11A haploinsufficiency

AbstractThe BCL11A gene encodes a transcriptional repressor with essential functions in multiple tissues during human development. Haploinsufficiency for BCL11A causes Dias-Logan syndrome (OMIM 617101), an intellectual developmental disorder with hereditary persistence of fetal hemoglobin (HPFH). Due to the severe phenotype, disease-causing variants in BCL11A occur de novo. We describe a patient with a de novo heterozygous variant, c.1453G>T, in the BCL11A gene, resulting in truncation of the BCL11A-XL protein (p.Glu485X). The truncated protein lacks the 3 C-terminal DNA-binding zinc fingers and the nuclear localization signal, rendering it inactive. The patient displayed high fetal hemoglobin (HbF) levels (12.1-18.7% of total hemoglobin), in contrast to the parents who had HbF levels of 0.3%. We used cultures of patient-derived erythroid progenitors to determine changes in gene expression and chromatin accessibility. In addition, we investigated DNA methylation of the promoters of the γ-globin genes HBG1 and HBG2. HUDEP1 and HUDEP2 cells were used as models for fetal and adult human erythropoiesis, respectively. Similar to HUDEP1 cells, the patient's cells displayed Assay for Transposase-Accessible Chromatin (ATAC) peaks at the HBG1/2 promoters and significant expression of HBG1/2 genes. In contrast, HBG1/2 promoter methylation and genome-wide gene expression profiling were consistent with normal adult erythropoiesis. We conclude that HPFH is the major erythroid phenotype of constitutive BCL11A haploinsufficiency. Given the essential functions of BCL11A in other hematopoietic lineages and the neuronal system, erythroid-specific targeting of the BCL11A gene has been proposed for reactivation of γ-globin expression in β-hemoglobinopathy patients. Our data strongly support this approach.

THALASSEMIA SYNDROME AND OTHER HEMOGLOBINOPATHIES: A SINGLE THALASSEMIA CENTRE EXPERIENCE FROM TERTIARY CARE HOSPITAL OF PURULIA

Background: Hemoglobinopathies are one of the major health burden globally including India and Southeast Asian region. This study was carried out to nd out the prevalence of β-thalassemia, HbS, HbD, HbE as well as identication of asymptomatic carriers who have an increased risk of having a child with thalassemia in the western part of West Bengal. Materials and methods: In this retrospective cohort study, the data was collected from all newly registered patients with anemia, referred from different outdoor patients in a tertiary care hospital, attending thalassemia clinic over a period of 3 years from January2018 to December2020. Detailed clinical history was taken and blood samples collected are tested with Sysmex automated blood cell counter for red cell indices. Diagnosis of hemoglobinopathy was done by G8 HPLC (high performance liquid chromatography) Analyzer by TOSOH Bioscience. Results: A total 2297 cases were studied during the study period. The age of the patients ranged between 6 months to 60 years. Higher percentage of the study sample was at age of more than 10 years (93.8%). Among 2297 cases, 477 cases showed abnormal Hb fractions on HPLC. The following Hb abnormalities detected were following: β (beta) thalassemia trait 13.2%, β thalassemia major 2.3% followed by sickle cell trait 2.17%, HbE trait 1.8%, HbS disease 0.21%, Hb E disease 0.13%, hereditary persistence of fetal hemoglobin (HPFH) traits 0.17% and Hb D trait 0.08% along with HbE-β-thalassemia 0.30% and HbS-β-thalassemia 0.26%. Conclusion: Among the hemoglobinopathies, β-thalassemia trait (12.9%) is prevalent in western part of Bengal.

Characterization of β-Globin Gene Cluster Deletions Using Multiplex-Gap Polymerase Chain Reaction (PCR) and Multiplex Ligation-Dependent Probe Amplification (MLPA)

Background : Deletions in the β-globin gene cluster are usually rare, problematic to detect, and subsequently possess a challenge in many diagnostic laboratories. They are normally related to the heterozygous of the delta beta (δβ) thalassemia, hereditary persistence of fetalhemoglobin (HPFH) and some of the hemoglobin variants. These disorders are typically presented by elevated levels of hemoglobin F (Hb F), but with low to normal hemoglobin A2 (Hb A2). However, despite their existence, there is still a limited number of studies focusing on this area, hence no definitive diagnosis could be conclusively established. Therefore, this pilot study was carried out to fill this knowledge gap.
 Methods: In this study, screening of the selected deletional mutations in the β-globin gene cluster among patients with Hb F (>1%) and Hb A2 (<4%) were performed using multiplex Gap-PCR and multiplex ligation-dependent probe amplification (MLPA).
 Results: The results showed that out of 54 samples tested using multiplex Gap-PCR against four target deletions; Thai (δβ)°-thalassemia, HPFH-6, Siriraj J and HbLepore, one sample was found positive with Thai (δβ)°-thalassemia. Further findings from the MLPA screening on 12 randomly selected samples revealed that another patient was positive with double deletions within the region of the β-globin gene cluster. These deletions occur at the gamma-globin gene 1 (HBG1) and gamma-globin gene 2 (HBG2) in exon 3.
 Conclusions: In conclusion, this study highlighted the importance of these deletions’ characterization using multiplex Gap-PCR and MLPA which helps in establishing a definitive diagnosis among this selected group of patients.
 Bangladesh Journal of Medical Science Vol.20(3) 2021 p.618-624

Physiological and Aberrant γ-Globin Transcription During Development.

The expression of the fetal Gγ- and Aγ-globin genes in normal development is confined to the fetal period, where two γ-globin chains assemble with two α-globin chains to form α2γ2 tetramers (HbF). HbF sustains oxygen delivery to tissues until birth, when β-globin replaces γ-globin, leading to the formation of α2β2 tetramers (HbA). However, in different benign and pathological conditions, HbF is expressed in adult cells, as it happens in the hereditary persistence of fetal hemoglobin, in anemias and in some leukemias. The molecular basis of γ-globin differential expression in the fetus and of its inappropriate activation in adult cells is largely unknown, although in recent years, a few transcription factors involved in this process have been identified. The recent discovery that fetal cells can persist to adulthood and contribute to disease raises the possibility that postnatal γ-globin expression could, in some cases, represent the signature of the fetal cellular origin.

In vivo HSPC gene therapy with base editors allows for efficient reactivation of fetal γ-globin in β-YAC mice

AbstractBase editors are capable of installing precise genomic alterations without creating double-strand DNA breaks. In this study, we targeted critical motifs regulating γ-globin reactivation with base editors delivered via HDAd5/35++ vectors. Through optimized design, we successfully produced a panel of cytidine and adenine base editor (ABE) vectors targeting the erythroid BCL11A enhancer or recreating naturally occurring hereditary persistence of fetal hemoglobin (HPFH) mutations in the HBG1/2 promoter. All 5 tested vectors efficiently installed target base conversion and led to γ-globin reactivation in human erythroid progenitor cells. We observed ~23% γ-globin protein production over β-globin, when using an ABE vector (HDAd-ABE-sgHBG-2) specific to the –113A>G HPFH mutation. In a β-YAC mouse model, in vivo hematopoietic progenitor/stem cell (HSPC) transduction with HDAd-ABE-sgHBG-2 followed by in vivo selection resulted in >40% γ-globin+ erythrocytes in the peripheral blood. This result corresponded to 21% γ-globin production over human β-globin. The average –113A>G conversion in total bone marrow cells was 20%. No alterations in hematological parameters, erythropoiesis, and bone marrow cellular composition were observed after treatment. No detectable editing was found at top-scoring, off-target genomic sites. Bone marrow lineage–negative cells from primary mice were capable of reconstituting secondary transplant-recipient mice with stable γ-globin expression. Importantly, the advantage of base editing over CRISPR/Cas9 was reflected by the markedly lower rates of intergenic HBG1/2 deletion and the absence of detectable toxicity in human CD34+ cells. Our observations suggest that HDAd-vectorized base editors represent a promising strategy for precise in vivo genome engineering for the treatment of β-hemoglobinopathies.

β-globin gene cluster mutations and large deletions among anaemic patients with elevated HbF level in a tertiary teaching hospital in Kelantan, Malaysia

Mutations in the β-globin gene cluster can lead to β-thalassaemia, δβ-thalassaemia, hereditary persistence of foetal haemoglobin (HPFH) and some of the haemoglobin variants. The clinical and haematological spectrum of thalassaemia range from benign to pathogenic conditions including severe transfusion dependent thalassaemia. Therefore, it is important to accurately diagnose β-globin gene cluster mutations to prevent thalassaemia major or intermedia offspring. The objective of this study is to detect β-globin gene cluster mutations and large deletions among anaemic patients with elevated HbF level in a tertiary teaching hospital in Kelantan, Malaysia. This study involved 144 anaemic patients with HbF level >1.0%. High-Performance Liquid Chromatography (HPLC) was used to determine the HbF and HbA2 levels. Multiplex-ARMS (ARMS)-PCR and gap-PCR were performed for those patients with high HbA2 level (>3.2%) and normal HbA2 level (≤3.2%) to detect β-globin gene cluster mutations and large deletions respectively. The majority of patients were Malays (99.3%) with mean age of 19.99 ± 1.64 years and female 61.1% predominance. Out of 87 samples tested using multiplex ARMS-PCR against eight targeted single mutation; Cd 41/42, IVS 1–5, Cd 26, Cd 17, Cd 71/72, IVS 1–1, Cd 8/9 and -28 mutations, 65 (74.7%) patients were detected which comprises of Cd 26 (56.3%), Cd 41/42 (11.5%), compound Cd 26 and Cd 41/42 (3.4%) and IVS 1–1 (3.4%). Meanwhile, for multiplex gap-PCR which detect four types of large deletions; Thai (δβ)o-thalassaemia, HPFH-6, Siriraj J and Hb Lepore, one out of 57 patients (1.8%) was found positive with Thai (δβ)o-thalassaemia. There was a significant difference between the mean of HbF level, MCV level as well as MCH level of patients with and without β-globin gene mutations and large deletions (P<0.05). This study highlighted the presence of various types of β-globin gene cluster mutations detection in establishing a definitive diagnosis among this selected group of patients for the large-scale screening of the thalassaemia gene.

Manipulation of Developmental Gamma-Globin Gene Expression: an Approach for Healing Hemoglobinopathies.

β-Hemoglobinopathies are the most common monogenic disorders, and a century of research has provided us with a better understanding of the attributes of these diseases. Allogenic stem cell transplantation was the only potentially curative option available for these diseases until the discovery of gene therapy. The findings on the protective nature of fetal hemoglobin in sickle cell disease (SCD) and thalassemia patients carrying hereditary persistence of fetal hemoglobin (HPFH) mutations has given us the best evidence that the cure for β-hemoglobinopathies remains hidden in the hemoglobin locus. The detailed understanding of the developmental gene regulation of gamma-globin (γ-globin) and the emergence of gene manipulation strategies offer us the opportunity for developing a γ-globin gene-modified autologous stem cell transplantation therapy. In this review, we summarize different therapeutic strategies that reactivate fetal hemoglobin for the gene therapy of β-hemoglobinopathies.

Spectrum of hemoglobin disorders in southern Odisha, India: a hospital based study.

Background:Hemoglobin disorders are the leading health concern in the world including India. There is a paucity of literature on the spectrum of hemoglobin disorders in southern districts of Odisha state. This study was undertaken to elucidate the occurrence of different hemoglobin disorders in a tertiary health care facility of Odisha state, India.Methods:The study cases were suspected patients of all age groups advised for screening of different hemoglobin disorders. Hemoglobin disorders were screened by sickling slide test and high-performance liquid chromatography (HPLC) using the Variant-II hemoglobin testing system as per the manufacturer's guidelines.Results:Over 2 years, 2332 blood samples (including 1102 pediatric and 1230 adult cases) were investigated, out of which, 1380 (59.2%) of cases had abnormal hemoglobin disorders. The most common was sickle cell disorders (48.67%, 1135/2332) followed by β-thalassemia (11.32%, 264/2332). Some rare variants were detected as hemoglobin D-Punjab, hemoglobin E, hemoglobin Lepore, hereditary persistence of fetal hemoglobin, hemoglobin with high P2 window, hemoglobin with high P3 window etc, Among the cases with abnormal hemoglobin disorders, 744 (53.9%), 545 (39.5%) and, 91 (6.6%) cases were found to have the heterozygous, homozygous and, double heterozygous state. Of the 188 ante-natal cases screened, 31.4% of cases had abnormal hemoglobin variants with sickle cell disorders being the most prevalent one.Conclusion:Along with the high occurrence of sickle cell disorders in the study area, some other rare hemoglobin disorders are also prevalent which calls for a large community-based cohort study.

Genetic Heterogeneity in Heterocellular Hereditary Persistence of Fetal Hemoglobin

AbstractA large English pedigree in which heterocellular hereditary persistence of fetal hemoglobin (HPFH) segregates is described. β-globin cluster deletions and γ gene promoter mutations associated with HPFH have been excluded. Of particular importance in this pedigree is the absence of any cosegregating hemoglobinopathy, thus allowing observation of the segregation pattern of this form of HPFH without the complicating effect of a β-globin gene mutation. Information gained in this study confirms that the extent of elevation of hemoglobin (Hb) F and F cells varies between affected individuals. There are one example of incomplete penetrance and three examples of father-to-son transmission, thus excluding X-linked inheritance. Consistent with previous reports, the most likely mode of inheritance is autosomal codominant. Linkage studies using a β-globin cluster microsatellite show no evidence of linkage to this chromosomal region implicating the presence of trans-acting regulatory factor(s). We have recently mapped one such locus to the chromosome 6q region in a very large Asian-Indian pedigree. Linkage to chromosome 6q in the English pedigree was excluded, thus indicating the presence of genetic heterogeneity in heterocellular HPFH.

β-Hemoglobinopathies: The Test Bench for Genome Editing-Based Therapeutic Strategies.

Hemoglobin is a tetrameric protein composed of two α and two β chains, each containing a heme group that reversibly binds oxygen. The composition of hemoglobin changes during development in order to fulfill the need of the growing organism, stably maintaining a balanced production of α-like and β-like chains in a 1:1 ratio. Adult hemoglobin (HbA) is composed of two α and two β subunits (α2β2 tetramer), whereas fetal hemoglobin (HbF) is composed of two γ and two α subunits (α2γ2 tetramer). Qualitative or quantitative defects in β-globin production cause two of the most common monogenic-inherited disorders: β-thalassemia and sickle cell disease. The high frequency of these diseases and the relative accessibility of hematopoietic stem cells make them an ideal candidate for therapeutic interventions based on genome editing. These strategies move in two directions: the correction of the disease-causing mutation and the reactivation of the expression of HbF in adult cells, in the attempt to recreate the effect of hereditary persistence of fetal hemoglobin (HPFH) natural mutations, which mitigate the severity of β-hemoglobinopathies. Both lines of research rely on the knowledge gained so far on the regulatory mechanisms controlling the differential expression of globin genes during development.

Alpha chain inclusions in peripheral blood erythroblasts and erythrocytes

Because of the imbalance between the rate of synthesis of α and β globin chains, α chains precipitate within erythroblasts in the bone marrow in β thalassemia intermedia and major. Sometimes they are also apparent in the peripheral blood in both erythroblasts and erythrocytes. They are particularly likely to be seen in erythrocytes if the pitting function of the spleen has been lost as a result of splenectomy. The top images (×100 objective) show the peripheral blood film of a young woman with β thalassemia major who has had a splenectomy and who, because of psychological problems, has been receiving suboptimal transfusion therapy. Her blood count showed: total nucleated cell count 185 × 109/L, WBC 7.4 × 109/L, Hb 63 g/L, MCV 68 fL, MCH 20 pg and MCHC 291 g/L. The blood film is dimorphic (post-transfusion) and in addition shows nucleated red blood cells (two with lobulated nuclei), Howell-Jolly bodies, Pappenheimer bodies and an acanthocyte. There are three erythrocytes containing α chain inclusions (arrows) [top left and right image]. The lower images are from a patient with β thalassemia intermedia (also known as non-transfusion-dependent β thalassemia) with an unusual molecular mechanism. This 10-year-old African patient had compound heterozygosity for β0 thalassemia and hereditary persistence of fetal hemoglobin (HPFH2). The blood count showed Hb 94 g/L, MCV 62 fl, MCH 18.5 pg and MCHC 297 g/L. The blood film (lower images, ×100) shows anisocytosis, poikilocytosis, hypochromic cells and Pappenheimer bodies. There are two erythroblasts, both of which contain an α chain inclusion. In the left image the cytoplasm surrounding the inclusion is packed with Pappenheimer bodies [bottom left and right image]. The presence of α chain inclusions in these thalassemic conditions provides visual evidence of severe chain imbalance, both cases having a total lack of β chain synthesis with the condition in the second patient being only partly ameliorated by increased γ chain synthesis. Nothing to report. Not applicable