Hereditary Persistence Of HbF Research Articles

Development of Best-in-Class Gene Editing Therapy for β-Hemoglobinopathies Using Innovative Transformer Base Editor (tBE)

β-thalassemia and sickle cell disease (SCD) are among the most prevalent monogenic disorders worldwide and both these β-hemoglobinopathies are caused by mutations in the β-globin gene HBB. Reactivating the expression of the γ-globin genes ( HBG1/2) mimicking the naturally occurring hereditary persistence of HbF (HPFH) is expected to be a universal strategy to treat β-thalassemia and SCD by the induction of fetal hemoglobin (HbF). To achieve this goal, CorrectSequence Therapeutics' first pipeline, CS-101, uses transformer Base Editor (tBE) to precisely edit human hematopoietic stem cells (HSC) ex vivo to induce the expression of γ-globin for treatment of β-thalassemia and sickle cell disease. Compared with the commonly used gene editing methods such as CRISPR/Cas nucleases or other base editors, tBE is a base editing system that avoids to cause DNA double strand breaks or off-target mutations. With the safety advantages of tBE, CS-101 targets one of the most potent targets to activate γ-globin expression without causing unexpected off-target mutations. A commercial scale manufacturing process to ex vivo edit HSC has been developed and validated with more than 10 batches of commercial scale production, all of which exhibited consistent process performance and product quality. Pre-clinical in vivo studies showed that while tBE-mediated editing of HSC induces robust γ-globin expression, tBE did not cause adverse effect on the engraftment or differentiation of the HSC in mice after transplantation. Clinical study for CS-101 is underway and it holds great promise to become the best-in-class gene editing treatment for β-hemoglobinopathies.

DEVELOPMENT OF A SPIKE-IN FLOW-CYTOMETRY METHOD FOR THE QUANTITATION OF FETAL HEMOGLOBIN (HBF) IN INDIVIDUAL RED BLOOD CELLS OF SICKLE CELL DISEASE (SCD) PATIENTS

HbF is the most relevant modulator of the severity of SCD, inhibiting the polymerization of the mutant sickle hemoglobin (HbS). Despite its clinical relevance, a straightforward method to quantify and identify protective levels of HbF in individual red blood cells (RBCs) is still lacking. Here, we sought to develop a fluorescent-labeled RBC “Spike-In”(Spk) control (Ctr), to be mixed with patient samples, to serve as an internal reference in a novel quantitative flow-cytometry method. The Spk-Ctr was generated by mixing equal numbers of CFSE-labeled and fixed HbF Neg adult and HbF Pos cord blood (CB) RBCs, whose mean corpuscular HbF (MCHbF) mass was pre-calculated, based on the %HbF (HPLC-derived) and the MCH (hematology analyzer), using the formula: MCHbF=(%HbFxMCH)/100. Spk-Ctr was mixed with a similarly-fixed Test Sample, composed of 10% HbF Pos (prepared with the same RBCs as the Spk-Ctr), permeabilized, stained with an anti-HbF PE conjugate, and acquired by a flow cytometer. The anti-HbF PE Median Intensity Fluorescence (MIF) was obtained for gated HbF Pos RBCs in both the Ctr CFSE Pos and Test CFSE Neg gated populations. A conversion factor (CF) was calculated by dividing the pre-calculated MCHbF by the MIF obtained for HbF Pos RBCs from the CFSE Pos Spk-Ctr. By multiplying the anti-HbF PE fluorescence intensity of each RBC in the test sample by the CF, an absolute HbF/RBC values (in pg of HbF per RBC) can be obtained, which can be used to generate a distribution histogram to calculate the percentage of RBCs above a protective HbF/RBC threshold. The Ctr CB used had 73.2% HbF and a MCH of 36.4pg, resulting in a MCHbF of 26.64pg. The anti-HbF PE MIF of the Spk-Ctr HbF Pos RBCs was 25,166a.u. (arbitrary units), resulting in a CF of 0.001058. The anti-HbF PE MIF of the HbF Pos RBCs of the Test Sample was 24,146a.u., leading to an estimated MCHbF of 25.56pg; a deviation of only 4% from the expected (known) MCHbF. In contrast, when samples were permeabilized and stained in separate tubes, this variation was 27%. Recently, a work described a laborious method requiring a standard curve, generated with rare samples from subjects with hereditary persistence of HbF, and calibration beads. This method correct only for experimental variations arising from flow-cytometry acquisition and bath-to-bath antibody affinity/fluorescence differences, but not from experimental variability associated with sample handling of the test patient samples and those used in the standard curve. Our method eliminates the complexity associated with the use of a cumbersome standard curve, as the Spike-In Ctr and patient cells are subjected to the exact same procedures (in the same tube). Thus, experimental variability (derived from differences in, pipetting, incubation time, temperature, affinity/fluorescence of antibody batches, instrument or acquisition settings, etc.) is not expected to interfere with the quantitation. Once fully developed and validated, our method may serve as an auxiliary tool for the clinical management of SCD patients, contributing to the prediction of complications (allowing preventive measures to be adopted), or allowing the monitoring of the response to therapeutic approaches based on the induction of HbF (including hydroxyurea).

Delta beta thalassemia, a rare hemoglobin variant: An experience from nodal centre in North Indian state

CONTEXT: Fetal hemoglobin (HbF) reduces to <1% few months after birth. However, it can persist beyond infancy into adult life in rare conditions such as delta-beta (δβ) thalassemia and hereditary persistence of HbF. δβ thalassemia is a relatively rare type of thalassemia due to decrease in both beta and delta globin chain production.AIMS: This study aims to assess the hematological and high-performance liquid chromatography (HPLC) findings in δβ thalassemia.SETTINGS AND DESIGN: Cross-sectional observational study.SUBJECTS AND METHODS: The study was conducted in department of Clinical Pathology, PGIMS, Rohtak. On screening of 1197 patients over a period of around 1 year, a total of 13 cases of δβ thalassemia were detected by HPLC.STATISTICAL ANALYSIS USED: Not done.RESULTS: The age of presentation ranged from 1 to 42 years with a mean age of 22.8 years. Eleven cases were diagnosed as heterozygous δβ thalassemia and two as homozygous δβ thalassemia.CONCLUSIONS: δβ thalassemia is an uncommon cause of elevated HbF. Clinical and hematological parameters should be carefully analyzed for accurate diagnosis.

Sickle Cell Disease and COVID-19 Infection: A Single-Center Experience

Background and Objectives:The COVID-19 (CO19) pandemic caused by SARS-CoV-2 remains a significant issue for global health, economics, and society. Several reports have shown that African Americans (AA) have been disproportionately affected by the CO19 pandemic. Limited data have suggested that sickle cell disease (SCD) could be one of the several reasons for higher morbidity and mortality related to CO19 among AA. Recent reports have suggested higher-than-average morbidity and mortality related to CO19 among patients with SCD. We conducted a retrospective, single-institution study in adult patients with SCD who were diagnosed with CO19 infection and their outcomes.Methods:After IRB approval, we conducted a chart review of adult patients (greater than 18 years) with SCD who were diagnosed with CO19 infection between March 1st, 2020, and March 31st, 2021. We recorded demographic data including age, gender, social factors (the type of insurance, availability of primary care provider (PCP), living alone/not), clinical parameters (type of SCD, co-morbidities), outpatient management of SCD, and how CO19 infection was managed like inpatient admission and complications. In patients who were admitted or seen in the emergency department (ED), we collected additional data including vitals, labs, the severity of illness, complications, length of stay, and outcomes. Computations were performed using statistical software SAS 9.4 for Windows.Results:We found a total of 51 patients with SCD diagnosed with CO19 infection in the above period. The median age of patients was 30 years. 61% were females and 39 % were males. All of them were AA. 11.76% were living alone, 49.02% were living with family, 1.96% (1 patient) was institutionalized, and the living situation was unknown in 37.25%. Most of the patients had Medicaid Insurance (52.94%), Medicare in 33.3%, private insurance in 13.73 % and 2% were uninsured. Only 64.71% of patients had a PCP.60% had HbSS disease, 32% had HbSC disease, 4% had HbS-beta thalassemia, one patient each had HbSS with hereditary persistence of HbF and HbS/HbD. Comorbidities and previous history included acute chest syndrome in 65.96%, avascular necrosis in 36.96%, leg ulcers in 8.7%, hypertension in 8.7%, sickle cell retinopathy in 14.57%, cerebrovascular disease in 26.19%, chronic kidney disease in 7.69%, venous thromboembolism (VTE) in 20.41%, 10.41% were on anticoagulation, history of HIV and hepatitis C infection in 6.38%. 28.21% of patients were maintained on partial exchange transfusions as an outpatient for various indications. 72.73% were on hydroxyurea, 7.5% were on crizanlizumab, 5.26% were on voxelotor and 26.83% were on iron chelation.Vitals and pertinent lab values on initial assessment were recorded and many patients had missing data. On presentation, 25.53% were febrile, 29.17% of patients were tachycardic, 31.25% were hypoxic (SpO2 < 95%), 38.46% were tachypneic, 59.18% had a body mass index (BMI) of > 24.9. Median hemoglobin and hematocrit were 8.9/27.4 g/dL. The median white blood cell count was 9490/uL and platelets were 315,000/uL. Median ferritin was 1573 ug/L. Median bilirubin and creatinine were 2.05 mg/dL and 0.86 mg/dL.The patients were further stratified based on the clinical location where CO19 infection was managed (Table 1). 39.3% were diagnosed in the outpatient setting/ED and 60.3% in the inpatient setting. Among 51 patients, 5.71% (n=2) required ICU admission and was mechanically ventilated. 17.5% received dexamethasone, 7.69% received remdesivir, 2.76% received convalescent plasma, 17.07% had infections and 47% received antibiotics. Only one patient received an exchange transfusion during admission. One patient developed a new VTE after CO19 infection. On statistical analysis, the only factor which impacted the clinical location of management was tachycardia (P=0.007).Of the 51 patients, only 3.9% (2 patients) died of complications of CO19 infection, one with hypoxic respiratory failure, disseminated intravascular coagulation, shock, and the other one with pulmonary embolism. 13% were readmitted within a month, one of them was admitted with a new pulmonary embolism and the others were admitted for acute painful episodes.Conclusion:We found a mortality rate of 3.9% in our single-center study of patients with SCD and CO19 infection. This mortality rate is lower than other published experiences in patients with SCD and CO19 infection. [Display omitted] DisclosuresMaster: Blue Bird Bio: Current holder of individual stocks in a privately-held company.

A New Tool for HbF Quantification Demonstrates That Hydroxyurea Decreases Mostly RBCs Containing Very Low Amounts of HbF

Introduction :Fetal hemoglobin (HbF) expression is a major modulator of sickle cell disease (SCD) severity. Hydroxyurea improves SCD clinical manifestations in part through its ability to induce HbF synthesis which decreases the HbS polymer formation. However, the distribution of HbF is heterogeneous in red blood cells (RBCs) from SCD patients. In the hypothesis of an HbF “threshold” for inhibiting the HbS polymerization, accurate measurement of the HbF content in RBC is an important issue. Several techniques have been developed to quantify HbF. Nevertheless, these technics are qualitative, or semi quantitative, allowing only to detect the RBCs containing a mild/high HbF level, called “F-cells”.Methods : we developed a simple and accurate method to quantify the HbF in each single RBCs and to evaluate the HbF distribution in SS RBCs during a 6 month Hydroxyurea therapy, as a proof of concept. RBCs adults carrying hereditary persistence of HbF (HPFH), β-Thalassemia or db-Thalassemia, were collected. Pancellular homogenous HbF distribution was controlled by flow cytometry. The mean amount of HbF (MCHbF) was calculated from the %HbF determined by HPLC and the mean corpuscular haemoglobin (MCH). The mean fluorescence intensity (MFI) after HbF staining by a monoclonal antibody was quantified by flow cytometry and normalized using quantitation beads.RBCs from homozygous SCD patients were collected before treatment (D0), between 15 days and 3 months (D15-M3) and after 6 months of HU treatment (M6). RBCs were frozen at -80°C. Non-inclusion criteria were vaso-occlusive crisis, blood transfusion within 3 months preceding the enrolment, and on-going pregnancy. After thawing, RBCs were analysed by flow cytometry after HbF staining.Results : Homogenous HbF distribution was proven by flow cytometry in 12 adults with HPFH, δβ thalassemia or β thalassemia. A linear regression between MCHbF and MFI, on 11 different experiments, was performed and provided an equation allowing the quantification of HbF (pg) by RBC (R²=0.94; p<0.0001). A mathematical model calculated the 95% confidence interval. Sixteen homozygous SCD adults were included for the study on HbF distribution under HU. RBCs containing 0 to 2 pg of HbF (table 1) decrease by 25% after 6 months of HU (p<0.0001) whereas a non-significant increase in the other categories of HbF by RBC was found.Conclusion : This simple technique, based on flow cytometry, could represent a new important tool in sickle cell and thalassemia diseases to better quantify the HbF expression and then better evaluate patient's pronostic and therapeutic effects. [Display omitted] DisclosuresGalactéros:Addmedica: Membership on an entity's Board of Directors or advisory committees. Bartolucci:Addmedica: Research Funding; GBT: Membership on an entity's Board of Directors or advisory committees; Fondation Fabre: Research Funding; Novartis US: Membership on an entity's Board of Directors or advisory committees.

A “Shot in the Arm” for Sickle Cell Disease

A “Shot in the Arm” for Sickle Cell Disease

Nationwide carrier detection and molecular characterization of \u03b2-thalassemia and hemoglobin E variants in Bangladeshi population

Backgroundß-thalassemia is one of the most common inherited blood disorders in the world and a major deterrent to the public health of Bangladesh. The management of thalassemia patients requires lifelong frequent blood transfusion and the available treatment options are unsatisfactory. A national policy on thalassemia prevention is mandatory in Bangladesh. However, precise and up-to-date information on the frequency of ß-thalassemia carriers are missing due to lack of accurate diagnostic approaches, limited access to information and absence of national screening program. This study aims to determine the nationwide carrier frequency of hemoglobin E (HbE) and β- thalassemia and mutation spectrum among the carriers using molecular, hematological and biochemical methods.MethodsThe study enrolled a total of 1877 individuals (60.1% male and 39.9% female) aged between 18 and 35 years. Total sample size and its division-wise breakdown were calculated in proportion to national and division-wise population. Venous blood was collected and subjected to CBC analysis and Hb-electrophoresis for each participant. Serum ferritin was measured to detect coexistence of iron deficiency anemia with thalassemia carrier. DNA-based High Resolution Melting (HRM) curve analysis was performed for confirmation of carrier status by mutation detection.ResultsOf 11.89% (95% CI, 10.43–13.35) carriers of β-globin gene mutations, 8.68% (95% CI, 7.41–9.95) had HbE trait (ETT) and 2.24% (95% CI, 1.57–2.91) had beta-thalassemia trait (BTT). Among eight divisions, Rangpur had the highest carrier frequency of 27.1% (ETT-25%, BTT-2.1%), whereas Khulna had the lowest frequency of 4.2% (ETT-4.2% only). Moreover, α- thalassemia, HbD trait, HbE disease, hereditary persistence of HbF were detected in 0.11, 0.16, 0.43 and 0.16% participants, respectively. HRM could identify two individuals with reported pathogenic mutations in both alleles who were erroneously interpreted as carriers by hematological indices. Finally, a total of nine different mutations including a novel mutation (c.151A > G) were detected in the β-globin gene.ConclusionsSince carrier frequency for both HbE and β-thalassemia is alarmingly high in Bangladesh, a nationwide awareness and prevention program should be made mandatory to halt the current deteriorating situations. Mutation-based confirmation is highly recommended for the inconclusive cases with conventional carrier screening methods to avoid any faulty detection of thalassemia carriers.

False positive testing for sickle hemoglobin in a blood donor with mild erythrocytosis and hemoglobin Geldrop St. Anna

Solubility testing for sickle hemoglobin is commonly performed to identify blood suitable for patients with sickle cell disease. A 32-year-old Caucasian male blood donor’s unit screened positive for sickle hemoglobin via solublity testing (Streck). As the donor was considered low risk for being positive for hemoglobin S (HbS), he self-referred to hematology for further evaluation. Testing with hemoglobin electrophoresis revealed the patient to be negative for HbS; however, 42 % fetal hemoglobin (HbF) was noted. Since this was higher than typically seen in hereditary persistence of HbF, deoxyribonucleic acid (DNA) sequencing of hemoglobin (Hb) was ordered through a referral laboratory. Hb gene sequencing revealed the patient to be heterozygous for Hb Geldrop St. Anna, a rare Hb variant. This variant has previously been shown to migrate in the HbF region with alkaline electrophoresis. The workup demonstrated that the oxygen dissociation curve was left-shifted consistent with slightly increased oxygen affinity of this variant. The patient’s hematocrits (Hct) from his past donations were 53 % and 54 % about two years apart and his Hct at his hematology evaluation was 53 %. This report describes the first case of Hb Geldrop St. Anna in the United States and was associated with a false positive HbS screen. This Hb variant is considered to be benign and has an increased oxygen affinity that is associated with mild erythrocytosis. The donor was allowed to continue donating blood products.

Fetal Hemoglobin Measurement per Red Blood Cell Provides Biological and Clinical Protective Thresholds

Introduction: Fetal hemoglobin (HbF) expression is a major modulator of sickle cell disease (SCD) severity by decreasing the HbS polymerization. However, the distribution of HbF in red blood cells (RBC) is heterogeneous in SCD patients. In the hypothesis of an HbF "threshold" in RBCs for inhibiting the HbS polymerization, accurate measurement of the HbF content in each red blood cell (HbF/RBC) is mandatory. To this purpose we developed a new and accurate method allowing the direct measurement of HbF content per RBC. Thanks to it, as a proof of concept, we analyzed HbF distribution and content in RBC from SCD patients before and after 6 months of treatment by hydroxyurea (HU). To determine if a threshold of HbF/RBC could modulate SCD, we analyzed the associations between the %RBC reaching different thresholds of HbF (in picograms), and biological parameters and the incidence of severe VOC. Methods: 14 SCD (βS/βS or βS/β0) patients were included to study HbF distribution during HU for a period of 6 months. RBCs were collected during each outpatient visit (Week 0, Week 2, Week 4, Week 12, and ≥ Week 24). HbF content was measured in RBCs using an anti-Human-HbF antibody by flow cytometry. Normalized RBC fluorescence intensity was then converted in picograms of HbF/RBC by using the linear association between mean HbF content and mean RBC fluorescence obtained from subjects presenting homogeneous HbF distribution (patients with hereditary persistence of HbF (HPFH), or β-Thalassemia or δβ-Thalassemia). Quantitative analysis were performed before and during HU treatment to characterize the response by comparing percentages of RBC classes based on different ranges of HbF/RBC during HU treatment. We therefore analyzed the associations between HbF/RBC thresholds (%RBC containing at least 2, 4, 6, 8, 10 or 20 pg HbF) and biological parameters before and ≥ 6 months of HU treatment. Finally HbF/RBC thresholds at Week 0 were compared to the incidence of hospitalized VOC within 3 years before W0, and HbF/RBC thresholds at Week 24 were compared to the incidence of hospitalized VOC within 3 years after W24 at a stable dose. Results: After 6 months of HU, mean %HbF, assessed by HPLC, raised from 6.16% (±3.5) to 15.2% (±8.7) (mean ± standard deviation). Quantitative analysis of HbF/RBC revealed a statistically significant decrease between D0 and ≥M6 of 24% of RBCs containing less than 2 pg (p = 0.0015) and a 2-fold increase of RBCs containing between 2 and 4 pg (p = 0.0025) (Friedman test). For biological parameters we observed an increase in mean %HbF, MCV and MCH and a decrease in RBC count significantly associated (p&lt;0.001 - Spearman test) with %RBC containing ≥ 2 pg of HbF. The incidence of VOC within 3 years after HU treatment was not statistically significant than during the 3 years before (p = 0.4414 - Wilcoxon test). VOC incidence under treatment decreased in 6/14 patients, did not change in 4/14 and increased in 4/14. The incidence of VOC over 3 years was not associated with the %HbF assessed by HPCL (r = -0.0358; p = 0.8564 - Spearman test). We observed a statistically significant correlation between the incidence of VOC over 3 years and the HbF threshold of 4 pg (r = -0.5068; p = 0.0059). We therefore determined the percentage of RBCs by thresholds of HbF, associated to ≤ 1 VOC over 3 years (Table 1). For example, if more than 20% RBCs have ≥ 4 pg of HbF, we calculated a sensitivity and a specificity of 58.3% and 100% respectively, and a positive and a negative predictive value of 100.0% and 76.2% respectively, to have ≤ 1 VOC over 3 years. Conclusion: Our results strengthen the hypothesis that the percentage of RBC above a threshold of HbF is the important parameter to measure. These results need to be replicated in a larger cohort but they open up interesting prospects for analysis of new therapeutic efficacy, including gene therapy and HbF inducers. Disclosures Galactéros: Addmedica: Membership on an entity's Board of Directors or advisory committees. Bartolucci:Agios: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; HEMANEXT: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; AddMedica: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Therapeutically relevant engraftment of a CRISPR-Cas9-edited HSC-enriched population with HbF reactivation in nonhuman primates.

Reactivation of fetal hemoglobin (HbF) is being pursued as a treatment strategy for hemoglobinopathies. Here, we evaluated the therapeutic potential of hematopoietic stem and progenitor cells (HSPCs) edited with the CRISPR-Cas9 nuclease platform to recapitulate naturally occurring mutations identified in individuals who express increased amounts of HbF, a condition known as hereditary persistence of HbF. CRISPR-Cas9 treatment and transplantation of HSPCs purified on the basis of surface expression of the CD34 receptor in a nonhuman primate (NHP) autologous transplantation model resulted in up to 30% engraftment of gene-edited cells for >1 year. Edited cells effectively and stably reactivated HbF, as evidenced by up to 18% HbF-expressing erythrocytes in peripheral blood. Similar results were obtained by editing highly enriched stem cells, defined by the markers CD34+CD90+CD45RA-, allowing for a 10-fold reduction in the number of transplanted target cells, thus considerably reducing the need for editing reagents. The frequency of engrafted, gene-edited cells persisting in vivo using this approach may be sufficient to ameliorate the phenotype for a number of genetic diseases.

Direct Promoter Repression by BCL11A Controls the Fetal to Adult Hemoglobin Switch

Fetal hemoglobin (HbF, α2γ2) level is genetically controlled and modifies severity of adult hemoglobin (HbA, α2β2) disorders, sickle cell disease, and β-thalassemia. Common genetic variation affects expression of BCL11A, a regulator of HbF silencing. To uncover how BCL11A supports the developmental switch from γ- to β- globin, we use a functional assay and protein binding microarray to establish a requirement for a zinc-finger cluster in BCL11A in repression and identify a preferred DNA recognition sequence. This motif appears in embryonic and fetal-expressed globin promoters and is duplicated in γ-globin promoters. The more distal of the duplicated motifs is mutated in individuals with hereditary persistence of HbF. Using the CUT&RUN approach to map protein binding sites in erythroid cells, we demonstrate BCL11A occupancy preferentially at the distal motif, which can be disrupted by editing the promoter. Ourfindings reveal that direct γ-globin gene promoter repression by BCL11A underlies hemoglobin switching.

The Prevalence and Role of Hemoglobin Variants in Biometric Screening of a Multiethnic Population: One Large Health System's Experience.

To characterize and quantitate hemoglobin (Hb) variants discovered during biometric hemoglobin A1c (HbA1c) analyses in a large multiethnic population with a focus on the effect of variants on testing method and results. In total, 13,913 individuals had their HbA1c measured via ion-exchange high-performance liquid chromatography. Samples that had a variant Hb detected or HbF fraction more than 25% underwent variant Hb characterization and confirmation by gel electrophoresis. RBC indices were also evaluated for possible concomitant thalassemia. Of the 13,913 individuals evaluated, 524 (3.77%) had an Hb variant. The prevalence of each variant was as follows: HbS trait (n = 396, 2.85%), HbSS disease (n = 4, 0.03%), HbC trait (n = 85, 0.61%), HbCC disease (n = 2, 0.01%), HbSC disease (n = 5, 0.04%), HbE trait (n = 18, 0.13%), HbD or G trait (n = 9, 0.06%), HbS β-thalassemia + disease (n = 1, 0.01%), hereditary persistence of HbF (n = 2, 0.01%), and HbMontgomery trait (n = 1, 0.01%). Concomitant α-thalassemia was detected in 20 (3.82%) of the 524 individuals with an Hb variant. This study represents one of the largest epidemiologic investigations into the prevalence of Hb variants in a North American metropolitan, multiethnic workforce and their dependents and reinforces the importance of method selection in populations with Hb variants.

758. A Nonhuman Primate Transplantation Model to Evaluate Gene Editing Strategies Aimed at Inducing Fetal Hemoglobin Production for the Treatment of Hemoglobinopathies

Fetal hemoglobin (HbF) is the major form of hemoglobin present in newborns but is almost completely replaced by adult hemoglobin after birth, where it constitutes less than 1 percent of total hemoglobin. Hereditary persistence of HbF is linked to mutations at multiple genetic loci that regulate the switch from fetal to adult hemoglobin. The targeting of these genes using site-specific nucleases thus constitutes a promising approach for the treatment of hemoglobinopathies by increasing HbF production.We have developed a nonhuman primate (NHP) model to investigate gene editing strategies aimed at inducing HbF production following hematopoietic stem cell (HSC) transplantation. As proof of principle, we focused on the transcription factor B-cell lymphoma/leukemia 11A (BCL11A), which functions as suppressor of HbF in humans. We disrupted the Bcl11a coding region using Transcription Activator-Like Effector Nucleases (TALENs) and achieved on average 30% gene editing by electroporation of mRNA in NHP CD34+ cells. Erythroid differentiation of these cells in culture confirmed that HbF expression was increased in Bcllla-edited cells as compared to control cells. To determine if Bcl11a-edited HSCs could engraft and give rise to HbF-producing erythrocytes, we transplanted a NHP with autologous CD34+ electroporated with Bcl11a TALEN mRNA following conditioning by total body irradiation. Using next generation sequencing, we detected about 1 % disruption in vivo one week after transplant, to reach a set point of about 0.3% over the course of the experiment. We were able to track several clones that persisted at least 200 days post transplantation based on their mutation signatures, suggesting engraftment of Bcllla-modified cells. HbF production was monitored in this animal by flow cytometry analysis of peripheral blood and was compared with three transplanted controls and one untransplanted control. In all transplanted animals, we observed a rapid increase in the frequency of F cells, reaching 10% to 40%, and lasting for about 140 days. In contrast, F cell production in the untransplanted control remained constant and minimal (<0.5%). After returning to basal levels, we found significantly higher HbF levels (1-1.5%) in the animal transplanted with Bcl11a-edited cells as compared to all other transplanted animals. These findings were confirmed by real-time PCR analysis of hemoglobin transcripts, which showed a 5-to 10-fold increase in gamma to beta globin ratio in the animal transplanted with Bcl11a-edited cells as compared to all controls. We also initiated work demonstrating the targeted integration of the chemoselection cassette P140K/MGMT at the Bcl11a locus in NHP HSCs by co-delivery of TALEN mRNA with a donor template carried on an adeno-associated viral vector, offering the potential for in vivo selection of modified cells. In summary, our experiments establish the NHP as pre-clinical model to evaluate therapeutic gene editing strategies for the treatment of hemoglobinopathies.

High Foetal Haemoglobin in Sickle Cell Disease: Not so Protective?

High Foetal Haemoglobin in Sickle Cell Disease: Not so Protective?

Fetal Hemoglobin In Sickle Cell Anemia: A Glass Half Full?

HbF modulates the phenotype of sickle cell anemia by inhibiting deoxyHbS polymerization. HbF is confined to erythrocytes called F-cells that can be detected by FACS when these cells contain sufficient HbF. Measuring the amount of HbF/F-cell is difficult and not clinically available. African-Americans with sickle cell anemia have 2-80% F-cells with an average HbF/F-cell of 6.4±1.6 pg. The distribution of HbF/F-cell is highly individual regardless of HbF level. People with HbS-gene deletion hereditary persistence of HbF (HPFH) have a mean HbF of 30%, and HbF is evenly distributed among their erythrocytes. Polymer is not present in these cells either experimentally or after calculating the HbS polymer fraction at 70% O2 saturation. Therefore, each cell contains about 10 pg. of HbF. DeoxyHbS polymerization is prevented at physiologic venous and capillary O2 saturations of 40-70% when HbF/F-cell is 9-12 pgs. We call this the “protective” level of HbF. F-cells need not contain “protective” levels of HbF. Some β-globin gene cluster haplotypes are associated with high HbF. Carriers of these haplotypes can have milder disease. Nevertheless, even patients with high HbF can have frequent painful episodes, acute chest syndrome and osteonecrosis. Patients with HbS-δβ thalassemia have 15 to 25% HbF but are anemic and have vasoocclusive complications, albeit less often than in sickle cell anemia. Hydroxyurea reduces the morbidity and mortality of sickle cell anemia, an effect likely to be mediated by its induction of HbF. Patients treated with hydroxyurea are better and probably live longer, but adults are anemic and rarely asymptomatic. In all these patient groups, HbF is unevenly distributed among erythrocytes. In contrast, people with HbS-HPFH are nearly asymptomatic and not anemic. The failure of HbF to modulate uniformly all complications of sickle cell disease might be related to the heterogeneous concentration of HbF in sickle erythrocytes. HbF is associated with protection from the development of certain disease subphenotypes but has limited prognostic value in individuals. In many cross-sectional studies, high HbF was associated with a reduced rate of acute painful episodes, fewer leg ulcers, less osteonecrosis, less frequent acute chest syndromes and reduced disease severity. HbF had a weak or no clear association with priapism, urine albumin excretion, stroke and silent cerebral infarction, systemic blood pressure and tricuspid regurgitant velocity. Perhaps this is because intravascular hemolysis of cells with little or no HbF causes nitric oxide scavenging, or because these complications are less dependent on HbS polymerization. No study provides information on the concentration of HbF/F-cell other than providing the relatively meaningless calculated mean value. Rather than the total number of F-cells or the concentration of HbF in the hemolysate, HbF/F-cell and the proportion of F-cells that have “protective” HbF is the most critical predictor of the likelihood of some disease subphenotypes. Hypothetical distributions of HbF-cells with different levels of HbF/F-cell can be plotted for different concentrations of HbF. With mean HbF levels of 5%, 10% and 20%, and HbF content per cell of 1.5, 3 and 6 pg., assuming a fixed mean, the variance was changed to show how the distribution of HbF per cell can greatly vary, even if the mean is constant. For example, with 20% HbF, as few as 1% and as many as 24% of cells have “protective” HbF. When HbF is lower, few or no “protected” cells can be present. Due to the heterogeneous concentrations of HbF, HbS can polymerize in some F-cells that have sub-polymer inhibiting concentrations of HbF. Inducing high levels of HbF is one approach to treating sickle cell disease. Inactivating BCL11A, a repressor of γ-globin gene expression, abrogates sickle cell disease in transgenic sickle mice. Their HbF was distributed homogeneously, and their phenotype mimicked HbS-HPFH. If it becomes possible in humans to target BCL11A or its pathway with agents that affect gene transcription, will it result in pancellular HbF? Broadening the distribution of HbF amongst sickle erythrocytes with drugs like hydroxyurea that effect the kinetics of erythropoiesis, coupled with an agent whose primary mechanism of action is to increase the transcription of the γ-globin genes, might be the most fruitful approach to HbF induction therapy and more efficacious than single agent treatment. Disclosures: No relevant conflicts of interest to declare.

Erythroid transcription factor EKLF/KLF1 mutation causing congenital dyserythropoietic anemia type IV in a patient of Taiwanese origin: Review of all reported cases and development of a clinical diagnostic paradigm

KLF1 is an erythroid specific transcription factor that is involved in erythroid lineage commitment, globin switching and terminal red blood cell maturation. Various mutations of KLF1 have been identified in humans, which have led to both benign and pathological phenotypes. The E325K mutation, within the second zinc finger of the KLF1 gene, has been shown to cause a new form of congenital dyserythropoietic anemia (CDA) now labeled as CDA type IV. We report the fourth documented case of this mutation, and propose a clinical diagnostic model to better identify this disease in other patients. Our patient is a Taiwanese child who presented to us at 8years of age with severe hemolytic anemia, splenomegaly, elevated fetal hemoglobin (HbF), iron overload, and dyserythropoiesis in the bone marrow. KLF1 sequence analysis revealed a G-to-A transition in one allele of exon 3, which resulted in the substitution of a glutamate 325 by a lysine. Flow cytometry analysis revealed decreased protein expression of CD44 on the red blood cells, and decreased red blood cell deformability as measured using an ektacytometer. Blood typing revealed his red blood cells to be Co(a-b-), In(b-), LW(ab-) and Lu(b+), even though DNA testing predicted that he would be Co(a+b-) and LW(a+b-). This newly discovered CDA combines features of a hemoglobinopathy, RBC membrane defect and hereditary persistence of HbF (HPFH) which are not seen in the previous types of CDA. Increased awareness of this phenotype may improve the more prompt and accurate diagnosis of these patients.

Issue Highlights—January 2013

Issue Highlights—January 2013

Therapeutic levels of fetal hemoglobin in erythroid progeny of β-thalassemic CD34+ cells after lentiviral vector-mediated gene transfer

Abstractβ-Thalassemia major results from severely reduced or absent expression of the β-chain of adult hemoglobin (α2β2;HbA). Increased levels of fetal hemoglobin (α2γ2;HbF), such as occurs with hereditary persistence of HbF, ameliorate the severity of β-thalassemia, raising the potential for genetic therapy directed at enhancing HbF. We used an in vitro model of human erythropoiesis to assay for enhanced production of HbF after gene delivery into CD34+ cells obtained from mobilized peripheral blood of normal adults or steady-state bone marrow from patients with β-thalassemia major. Lentiviral vectors encoding (1) a human γ-globin gene with or without an insulator, (2) a synthetic zinc-finger transcription factor designed to interact with the γ-globin gene promoters, or (3) a short-hairpin RNA targeting the γ-globin gene repressor, BCL11A, were tested. Erythroid progeny of normal CD34+ cells demonstrated levels of HbF up to 21% per vector copy. For β-thalassemic CD34+ cells, similar gene transfer efficiencies achieved HbF production ranging from 45% to 60%, resulting in up to a 3-fold increase in the total cellular Hb content. These observations suggest that both lentiviral-mediated γ-globin gene addition and genetic reactivation of endogenous γ-globin genes have potential to provide therapeutic HbF levels to patients with β-globin deficiency.

Severe fetomaternal hemorrhage confirmed and quantified by flow cytometry using anti fetal hemoglobin antibodies

Severe fetomaternal hemorrhage (FMH) is a rare but potentially life-threatening perinatal complication (1). The traditional method for diagnosis of this situation is the acid elution (Kleihaur–Betke) test. However, this technique is relatively insensitive and non-specific, and does not allow quantification of the hemorrhagic process. We report a case of severe FMH confirmed and quantified by means of flow cytometric analysis using specific staining for cells containing fetal hemoglobin (HbF) in maternal circulation. We believe that this method is more sensitive, more specific and allows accurate quantification of FMH. A 30-year-old nullipara at 36 + 2 gestational weeks was admitted to the delivery room for decreased fetal movements. On admission, a sinusoidal fetal heart rate pattern was recorded (Fig. 1), and an immediate cesarean section was performed. A 2400 g female infant was delivered with Apgar scores of five and seven at 1 and 5 min, respectively. The infant was extremely pale, limp and bradycardic. Initial hematocrit and hemoglobin levels were 12.3% and 4.4 g/dL, respectively, while a direct Coombs' test was negative. She was treated initially by infusion of normal saline followed by packed red blood cells (20 cc/kg), until marked clinical improvement, and her hematocrit reached 35%. The rest of her postnatal course was unremarkable. Fetal cardiotogoram showing a sinusoidal fetal heart rate pattern. In an attempt to confirm the suspected diagnosis of FMH, we performed flow cytometric analysis of fetal erythrocytes in maternal blood samples obtained soon after delivery. The cells were washed, fixed permiabilized and stained with monoclonal antibodies against HbF according to the manufacturer's instructions (Bioatlantic, Nantes, France). Cell analysis was performed with a FACstarplus flow cytometer (Beckton-Dickenson, Mountain View, CA, USA). A 488-nm argon laser beam at 250 mV served for excitation. The threshold was set on forward light scatter to exclude platelets. Green fluorescence was measured using logarithmic amplification through a 530 ± 30 nm band-pass-filter. As a positive control we used 3 neonatal cord blood samples and as negative controls blood samples from five other women after normal deliveries. The positivity threshold was adjusted with positive controls. Almost 11% of maternal erythrocytes contained high HbF level, i.e. were of fetal origin (Fig. 2). These results were confirmed by analyzing the hemoglobin types in the hemolysate of the maternal blood using high performance liquid chromatography (results not shown). Histogram of flow cytometric analysis of three samples stained for HbF. The flourescence intensity is demonstrated on a log scale along the X-axis, and the cell count along the Y-axis. A. The presented case showing a major peak of HbF negative cells (maternal cells) and a minor peak of cells containing high levels of HbF, i.e, fetal cells. B. A sample of umbilical cord blood. C. A sample of control maternal blood. Detection of fetal cells in maternal circulation represents an important support for the clinical diagnosis of FMH. Most laboratories perform FMH estimates on the basis of the slide-based microscopic counting method of acid elution (Kleihaur–Betke) test. This technique is laborious and suffers from lack of sensitivity, subjectivity and precision (2). These limitations prompted the use of flow cytometry which overcomes all these limitations by rapid and sensitive cell-by-cell analysis (3). In the past, flow cytometry approaches of fetal cells in maternal blood relied mainly on detection of the human D antigen. However, this approach was applicable only to clinical situations with D antigen incompatibility and could not be utilized in all cases of FMH. Using antibodies to HbF as described in the current case allows for broad application to various clinical situations (4, 5). Moreover, the quantitative nature of flow cytometric analysis permits the distinction of true fetal cells, which contain HbF as the major form of hemoglobin, from maternal circulating F-cells that have lower cellular HbF content. This is particularly applicable for individuals with hereditary persistence of HbF or various hemoglobinopathies. To our knowledge, this is the first reported case of severe FMH confirmed and quantified by flow cytometry using anti HbF antibodies. Clinicians may wish to apply this rapid and accurate method for diagnostic purposes postnataly and even antenatally when clinical suspicion arises.

Detection of fetal red cells in fetomaternal hemorrhage using a fetal hemoglobin monoclonal antibody by flow cytometry.

The laboratory determination of the level of fetal cells in maternal circulation remains an important support in the obstetrical management of women with suspected uterine trauma and in the proper dose administration of anti-D for prevention of Rh hemolytic disease of the newborn. Limitations in the sensitivity and precision of the widely used manual Kleihauer-Betke test have prompted an increased utilization of flow cytometric methods for fetal cell detection in maternal blood samples. Murine monoclonal antibodies directed against fetal hemoglobin (HbF) were developed, conjugated to fluorescein isothiocyanate, and used in a multiparametric flow cytometric assay developed for the quantitation of fetal red cells. A rapid intracellular staining method using brief glutaraldehyde fixation and Triton X-100 permeabilization prior to monoclonal antibody incubation was developed, along with optimization of the flow cytometric analysis protocol for the analysis of 50,000 cells. The performance of the assay was assessed for linearity and precision and correlated with the Kleihauer-Betke acid elution method. The anti-HbF flow cytometric method showed good correlation with the Kleihauer-Betke method (r2 = 0.86) and superior precision with a CV < 15 percent for blood samples with > 0.1 percent fetal cells. Analysis of 150 blood samples from nonpregnant adults, including individuals with elevated HbF due to hemoglobinopathies and hereditary persistence of HbF, gave a mean value of 0.02 percent fetal cells, and all results were less than 0.1 percent. The anti-HbF flow cytometric method for detection of fetal cells offers a simple, reliable, and more precise alternative to the Kleihauer-Betke manual technique for the assessment of fetomaternal hemorrhage. The method has additional potential applications for the study of HbF levels or frequency of adult red cells with low levels of HbF (F cells) in individuals with hemoglobinopathies.