Mutant Hemoglobin Research Articles

Ten years of a neonatal screening program for hemoglobinopathies in Friuli-Venezia Giulia: first regional experience in Italy.

Hemoglobinopathies are the commonest genetic defect worldwide (7% of the world's population has at least one hemoglobin mutation). Although prenatal screening for hemoglobinopathies is not obligatory during pregnancy in Italy, it is offered to women by the Italian National Health Service in the pre-conception phase. The screening of newborns is a valid alternative, and has been adopted in various European countries, albeit in a piecemeal fashion. Neonatal screening has the advantage of providing early diagnosis of a hemoglobinopathy. Here we report the findings from the experience with neonatal screening in Friuli-Venezia Giulia since 2010. The hemoglobinopathy screening project in Friuli-Venezia Giulia, a Region in north Italy, began in November 2010. High-performance liquid chromatography was performed on dried blood spot samples collected by obstetric nurses from neonates within 5-8 days after birth. From 2010 to 2019, 11,956 newborns were screened, and abnormal hemoglobin was found in 519 of them (4.34%): the variants identified included HbS, HbC, HbD, HbE and HbX. More specifically, the HbS variant was observed in 347 (2.9%) newborns and the homozygous pattern was identified in 24 (0.2%) cases. The screening also detected two cases of β-thalassemia major. We report our experience of 10 years of screening newborns for hemoglobinopathies in the Region of Friuli-Venezia Giulia, in which 7.7% of people come from malaria-endemic areas. Increased mobility and migratory flows bringing in hemoglobinopathy carriers from endemic areas have led to an increase in mutations in non-malarial countries, with a current incidence of around 4% in the newborns we tested. This means that hemoglobinopathies can be described as a rare condition. Our data show that incidence rates are comparable to those of other inherited disorders such as phenylketonuria, thereby justifying the inclusion of the test for hemoglobinopathies into screening programs for rare diseases.

Voxelotor (GBT440) in pediatric sickle cell disease: A review

Sickle cell disease (SCD) was first described in 1910 in African Americans, and the mutant hemoglobin S (HbS) was identified by electrophoresis in 1948. Sickle cell disease is the first genetic disease to be molecularly defined - a single point mutation in the β-globin gene (GAG→GTG) results in substitution of valine for glutamic acid at amino acid residue 7 (including the starting methionine). Pharmacological intervention to correct the defect at a molecular/protein level has proven complex. The only established curative therapy is hematopoietic stem cell transplantation, with recent gene therapy approvals providing hope for the same. Herein, we discuss voxelotor, a drug designed to reverse the hemoglobin polymerization defect caused by the β7Glu > Val substitution in the hemoglobin molecule.

In situ correction of various β-thalassemia mutations in human hematopoietic stem cells

β-thalassemia (β-thal) is the most common monogenic disorder caused by various mutations in the human hemoglobin β (HBB) gene and affecting millions of people worldwide. Electroporation of Cas9 and single-guide RNA (sgRNA)–ribonucleoprotein (RNP) complex-mediated gene targeting in patient-derived hematopoietic stem cells (HSCs), followed by autologous transplantation, holds the promise to cure patients lacking a compatible bone marrow donor. In this study, a universal gene correction method was devised to achieve in situ correction of most types of HBB mutations by using validated CRISPR/sgRNA–RNP complexes and recombinant adeno-associated viral 6 (rAAV6) donor-mediated homology-directed repair (HDR) in HSCs. The gene-edited HSCs exhibited multi-lineage formation abilities, and the expression of β-globin transcripts was restored in differentiated erythroid cells. The method was applied to efficiently correct different mutations in β-thal patient-derived HSCs, and the edited HSCs retained the ability to engraft into the bone marrow of immunodeficient NOD-scid-IL2Rg−/− (NSI) mice. This study provides an efficient and safe approach for targeting HSCs by HDR at the HBB locus, which provides a potential therapeutic approach for treating other types of monogenic diseases in patient-specific HSCs.

Sickle Cell Anemia Screening in Newborns and Analysis of Haplotypes in Patients from Santiago Island, Cape Verde.

Sickle cell anemia (SCA) results from a mutation in the β-globin gene, leading to the production of mutant hemoglobin, known as hemoglobin S (HbS). Despite being a genetic disorder, the phenotype of SCA can be influenced by the level of fetal hemoglobin (HbF), which is associated with beta S-globin haplotypes. In this study, we conducted newborn screening (NBS) using samples collected from umbilical cord blood in two hospitals on Santiago Island, Cape Verde. In newborns, HbS was detected using high-performance liquid chromatography (HPLC) on dried blood spot, with confirmation through polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). In addition, we assessed the hematological and clinical characteristics of a second population group consisting of patients diagnosed with SCA. Haplotype determination was performed on both newborns with HbS and patients with SCA. Beta S-globin haplotypes were determined using PCR-RFLP. Hematological values were analyzed using standard methods. Out of 346 newborns, 21 (6%) were carriers of the sickle cell trait (HbAS) while none were identified as homozygous for sickle cell disease (HbSS). Among both groups of individuals, four haplotypes were identified: Senegal, Arabi-Indian, Bantu, and Benin. The Senegal haplotype was the most prevalent, possibly reflecting the ethnic origin of the mutations observed. Hematological values did not differ significantly among haplotypes. However, higher levels of HbF were associated with better hematological values. These findings suggest a positive impact of elevated HbF levels on reducing the severity of SCA. Finally, we demonstrated how the combination of technics, HPLC and molecular analysis, provided a consistent and reproducible results that can be used for NBS for SCA.

Analysis of hemoglobin variants in Tianjin City and neighboring areas

To determine the types and proportion of common hemoglobin variants in Tianjin and surrounding areas, to analyze the recognition ability and the effects of hemoglobin variants on experimental results in two commonly used glycated hemoglobin systems, so as to provide data support for the consistency of HbA1c detection in Tianjin City. A case-control study was used for retrospective analysis,156 specimens with abnormal electrophoretic peaks in the detection of glycated hemoglobin were collected from more than 50 000 specimens of patients in Chu Hsien-I Memorial Hospital of Tianjin Medical University between June 2020 and December 2020. Determined their hemoglobin mutation sites by DNA sequencing, and compared the values of hemoglobin variants on glycated hemoglobin detection values by high performance liquid chromatography and capillary electrophoresis. SPSS 23 was used to calculate the blood routine results of the variant specimens, and compared with the normal reference interval. The results showed that DNA sequencing identified 21 hemoglobin variants, of which 11 were α strand variants and 10 were β strand variants. In addition, an unreported hemoglobin variant was identified, Hb Headington (HBB: c.217A>C). The HbA1c of 11 variants including Hb G-Honolulu, Hb Queens, Hb Q-Thailand, Hb J-Broussais, Hb O-Indonesia, Hb G-Coushatta, Hb G-Taipei, Hb E, Hb Headington, Hb New York and Hb D-Los Angeles were shifted by more than 7% when measured by high-performance liquid chromatography. Patients with the Hb Q-Thailand and Hb E cause reduced MCV and MCH. In conclusion, an unreported hemoglobin variant was found from Tianjin and neighboring areas. Patients with the Hb Q-Thailand and Hb E cause reduced MCV and MCH. 11 of these hemoglobin variants interfered with the detection of glycated hemoglobin using high-performance liquid chromatography, resulting in inaccurate results.

APOEe4*TREM2 and sickle cell trait in a late onset Alzheimer’s disease mouse model

AbstractBackgroundAlzheimer’s disease (AD) is a neurodegenerative disease associated with neuroinflammation, dysregulated neurovascular coupling, abnormal neuroplasticity, and neuropathological makers, including tau and β‐amyloid deposition. Advancing age is the greatest risk factor, and apolipoprotein E (APOE) e4 and triggering receptor expressed on myeloid cells 2 (TREM2) are genetic risk factors for late‐onset AD, which comprises >95% of all AD cases. Sickle cell trait (SCT) refers to the heterozygous inheritance of the sickle cell hemoglobin mutation. Studies from our laboratory and others show that SCT is associated with increased risk of microvascular complications and peripheral inflammation. Given the role of cerebral small vessel disease and inflammation in the pathogenesis of AD, we hypothesized that SCT may interact with late‐onset AD gene variants, resulting in earlier development of cognitive impairments, possibly via promoting neuroinflammation.MethodTo test our hypothesis, 10‐12 weeks old mice carrying APOE e4*TREM2 combined late‐onset AD genes were transplanted with bone marrow from humanized Townes mice with SCT (AS) or normal (AA) hemoglobin, following irradiation. Using a longitudinal cross‐sectional design, 3 cohorts were evaluated at 8, 12, and 18 months for presence of cognitive deficits using rodent behavioral testing. Mice were sacrificed immediately following behavioral testing, and brain tissue was evaluated using immunohistochemistry and Golgi‐Cox impregnation histology.ResultBehavioral testing suggested only age‐related cognitive impairments. There were no significant differences in neuroinflammation at any age between AS and AA mice in the hippocampus or corpus callosum and external capsule. Within genotypes, there was an age dependent increase in neuroinflammation, between 8 and 12 months in the hippocampus (p < 0.0001, p = 0.0002) and 12 versus 18 months in white matter tracts (p < 0.0001, p < 0.0001). There were no significant differences in the dendritic spine density or the proportion of immature spines between AS and AA mice. However, within genotypes there was significant differences between 12 and 18 months (p < 0.0001, p < 0.0001), with 18 months having lower spine density and higher proportion of immature spines.ConclusionAddition of SCT to a background of APOE e4*TREM2 did not result in earlier onset of cognitive impairment or more severe neuroinflammation.

How Do Sickle Cell Genes Protect Tribal People from Deadly Malaria? Is This a Type of Natural Selection?

In humans, genetic mutations have led to the formation of several haemoglobin (Hb) variants during human evolution. These mutations usually occur in the genes for a and Β- polypeptide or globin chains of haemoglobin located on chromosomes 11 and 16, respectively. Among these haemoglobin variants, sickle cell haemoglobin (Hb-S) is highly significant in the humans. In fact, this abnormal or mutant haemoglobin is the resultant of a point mutation in the gene for Β-globin, located on chromosome 11. No doubt, this sickle cell gene is relatively more prevalent among tribal people and widely distributed in those geographic regions where the dreaded Plasmodium falciparum-malaria is hyperendemic. In the human population, this mutant gene exists in two forms, the heterozygous (Hb-AS) and homozygous (Hb-SS) states. But this sickle cell gene is also found in association with other mutant genes of erythrocyte or red cell genetic disorders, such as abnormal Hb-C, Β-thalassemia, G-6-PD enzyme deficiency, etc. It is well known that tribal people generally live in forest ecosystem where dreaded malaria is also found to be endemic. How have these tribal people been able to protect themselves from this dreaded malaria for thousands of years in such a hostile environment, when they were most likely to be infected with the malaria parasite? Actually, its secret is hidden in these sickle cell genes only. Tribal individuals who have these genes in the heterozygous state (Hb-AS) do not get malaria or are protected from malaria and lead a healthy life. Several studies have confirmed that AS heterozygote people are protected against malaria. In contrast, those who have these genes in the homozygous (Hb-SS) state (homozygotes) develop sickle cell anaemia, which is fatal. Such patients die early due to severe malaria i.e AS heterozygotee individuals have a double advantages. In fact, it is a type of natural selection and also shows a balanced polymorphism in tribal people which is based on Darwin’s theory. This current communication provides factual and scientific information about the origin of sickle cell genes in the context of natural selection and how these genes protect tribal individuals from malaria. This communication is useful and important for zoologists, scientists working on human evolution, and medical scientists.

Effects of 2,3-DPG knockout on SCD phenotype in Townes SCD model mice.

Sickle cell disease (SCD) is a severe, multisystemic hematological disorder that impacts nearly every major organ in adults. The current approved treatments for SCD directly target mutant hemoglobin or address downstream disease pathology. Several compounds targeting reduction of 2,3-DPG by activation of Pyruvate Kinase-R are currently being evaluated in SCD patients. In this study, we genetically engineered a mouse lacking 2,3-DPG on the Townes SCD mouse model background and evaluated the effects of 2,3-DPG loss on disease pathology. Animals lacking 2,3-DPG showed improvements in hematological markers and reductions in RBC sickling relative to native Townes mice, however, minimal difference in organ damage was observed in 2,3-DPG deficient mice compared to native Townes animals. When animals lacking 2,3-DPG were dosed with a compound designed to increase hemoglobin oxygen affinity, oxygen delivery related toxicity was observed.

Exploring the host factors affecting asymptomatic Plasmodium falciparum infection: insights from a rural Burkina Faso study

BackgroundAsymptomatic Plasmodium falciparum parasitaemia forms a reservoir for the transmission of malaria disease in West Africa. Certain haemoglobin variants are known to protect against severe malaria infection. However, data on the potential roles of haemoglobin variants and nongenetic factors in asymptomatic malaria infection is scarce and controversial. Therefore, this study investigated the associations of iron homeostasis, inflammation, nutrition, and haemoglobin mutations with parasitaemia in an asymptomatic cohort from a P. falciparum-endemic region during the high transmission season.MethodsA sub-study population of 688 asymptomatic individuals (predominantly children and adolescents under 15 years, n = 516) from rural Burkina Faso previously recruited by the NOVAC trial (NCT03176719) between June and October 2017 was analysed. Parasitaemia was quantified with conventional haemocytometry. The haemoglobin genotype was determined by reverse hybridization assays targeting a selection of 21 HBA and 22 HBB mutations. Demographics, inflammatory markers (interleukins 6 and 10, hepcidin), nutritional status (mid upper-arm circumference and body mass index), and anaemia (total haemoglobin, ferritin, soluble transferrin receptor) were assessed as potential predictors through logistic regression.ResultsMalaria parasites were detected in 56% of subjects. Parasitaemia was associated most strongly with malnutrition. The effect size increased with malnutrition severity (OR = 6.26, CI95: 2.45–19.4, p < 0.001). Furthermore, statistically significant associations (p < 0.05) with age, cytokines, hepcidin and heterozygous haemoglobin S were observed.ConclusionsAccording to these findings, asymptomatic parasitaemia is attenuated by haemoglobin S, but not by any of the other detected genotypes. Aside from evidence for slight iron imbalance, overall undernutrition was found to predict parasitaemia; thus, further investigations are required to elucidate causality and inform strategies for interventions.

The dependence of maximum oxygen uptake and utilization (V̇O2 max) on hemoglobin-oxygen affinity and altitude.

Oxygen transport from the lungs to peripheral tissue is dependent on the affinity of hemoglobin for oxygen. Recent experimental data have suggested that the maximum human capacity for oxygen uptake and utilization (V̇O2 max) at sea level and altitude (~3000 m) is sensitive to alterations in hemoglobin-oxygen affinity. However, the effect of such alterations on V̇O2 max at extreme altitudes remains largely unknown due to the rarity of mutations affecting hemoglobin-oxygen affinity. This work uses a mathematical model that couples pulmonary oxygen uptake with systemic oxygen utilization under conditions of high metabolic demand to investigate the effect of hemoglobin-oxygen affinity on V̇O2 max as a function of altitude. The model includes the effects of both diffusive and convective limitations on oxygen transport. Pulmonary oxygen uptake is calculated using a spatially-distributed model that accounts for the effects of hematocrit and hemoglobin-oxygen affinity. Systemic oxygen utilization is calculated assuming Michaelis-Menten kinetics. The pulmonary and systemic model components are solved iteratively to compute predicted arterial and venous oxygen levels. Values of V̇O2 max are predicted for several values of hemoglobin-oxygen affinity and hemoglobin concentration based on data from humans with hemoglobin mutations. The model predicts that increased hemoglobin-oxygen affinity leads to increased V̇O2 max at altitudes above ~4500 m.

SMARCB1 regulates the hypoxic stress response in sickle cell trait

Renal medullary carcinoma (RMC) is an aggressive kidney cancer that almost exclusively develops in individuals with sickle cell trait (SCT) and is always characterized by loss of the tumor suppressor SMARCB1. Because renal ischemia induced by red blood cell sickling exacerbates chronic renal medullary hypoxia invivo, we investigated whether the loss of SMARCB1 confers a survival advantage under the setting of SCT. Hypoxic stress, which naturally occurs within the renal medulla, is elevated under the setting of SCT. Our findings showed that hypoxia-induced SMARCB1 degradation protected renal cells from hypoxic stress. SMARCB1 wild-type renal tumors exhibited lower levels of SMARCB1 and more aggressive growth in mice harboring the SCT mutation in human hemoglobin A (HbA) than in control mice harboring wild-type human HbA. Consistent with established clinical observations, SMARCB1-null renal tumors were refractory to hypoxia-inducing therapeutic inhibition of angiogenesis. Further, reconstitution of SMARCB1 restored renal tumor sensitivity to hypoxic stress invitro and invivo. Together, our results demonstrate a physiological role for SMARCB1 degradation in response to hypoxic stress, connect the renal medullary hypoxia induced by SCT with an increased risk of SMARCB1-negative RMC, and shed light into the mechanisms mediating the resistance of SMARCB1-null renal tumors against angiogenesis inhibition therapies.

Editing the core region in HPFH deletions alters fetal and adult globin expression for treatment of β-hemoglobinopathies

Reactivation of fetal hemoglobin (HbF) is a commonly adapted strategy to ameliorate β-hemoglobinopathies. However, the continued production of defective adult hemoglobin (HbA) limits HbF tetramer production affecting the therapeutic benefits. Here, we evaluated deletional hereditary persistence of fetal hemoglobin (HPFH) mutations and identified an 11-kb sequence, encompassing putative repressor region (PRR) to β-globin exon-1 (βE1), as the core deletion that ablates HbA and exhibits superior HbF production compared with HPFH or other well-established targets. PRR-βE1-edited hematopoietic stem and progenitor cells (HSPCs) retained their genome integrity and their engraftment potential to repopulate for long-term hematopoiesis in immunocompromised mice producing HbF positive cells invivo. Furthermore, PRR-βE1 gene editing is feasible without exvivo HSPC culture. Importantly, the editing induced therapeutically significant levels of HbF to reverse the phenotypes of both sickle cell disease and β-thalassemia major. These findings imply that PRR-βE1 gene editing of patient HSPCs could lead to improved therapeutic outcomes for β-hemoglobinopathy gene therapy.

Sickle Cell Hemoglobin Genotypes Affect Malaria Parasite Growth and Correlate with Exosomal miR-451a and let-7i-5p Levels.

Malaria affects a significant portion of the global population, with 247 million cases in 2021, primarily in Africa. However, certain hemoglobinopathies, such as sickle cell trait (SCT), have been linked to lower mortality rates in malaria patients. Hemoglobin (Hb) mutations, including HbS and HbC, can cause sickle cell disease (SCD) when both alleles are inherited (HbSS and HbSC). In SCT, one allele is inherited and paired with a normal allele (HbAS, HbAC). The high prevalence of these alleles in Africa may be attributed to their protective effect against malaria. Biomarkers are crucial for SCD and malaria diagnosis and prognosis. Studies indicate that miRNAs, specifically miR-451a and let-7i-5p, are differentially expressed in HbSS and HbAS compared to controls. Our research examined the levels of exosomal miR-451a and let-7i-5p in red blood cells (RBCs) and infected red blood cells (iRBCs) from multiple sickle Hb genotypes and their impact on parasite growth. We assessed exosomal miR-451a and let-7i-5p levels in vitro in RBC and iRBC supernatants. Exosomal miRNAs exhibited distinct expression patterns in iRBCs from individuals with different sickle Hb genotypes. Additionally, we discovered a correlation between let-7i-5p levels and trophozoite count. Exosomal miR-451a and let-7i-5p could modulate SCD and malaria severity and serve as potential biomarkers for malaria vaccines and therapies.

5613165 INVESTIGATING VARIANTS ASSOCIATED WITH QUANTITATIVE RED BLOOD CELL PHENOTYPES FROM TOPMED PREDICTIONS IN AFRICAN SICKLE CELL DISEASE POPULATIONS.

Background: Sickle cell disease (SCD) is caused by a single point mutation (c.20A>T, p.Glu6Val), which results in the formation of mutant haemoglobin S (HbS) instead of haemoglobin A (HbA). It is a life-long inherited recessive disorder that predominantly affects people of African, Indian, and Mediterranean descent. The polymerisation of HbS within red blood cells (RBCs) is the primary pathophysiological alteration in SCD, which results in the sickle-shape of RBCs. Sickle-shaped RBCs are more prone to haemolysis and blocking blood flow, resulting in anaemia and painful episodes. Haematological indices including haematocrit (HCT), haemoglobin concentration (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), red blood cell width (RDW) and RBC count indicate the size, development, and haemoglobin content of RBCs. Haematological indices are routinely measured as a means of monitoring SCD. A study recently found associations between 14 novel variants and haematological indices when using whole genome sequencing (WGS) data. The association between these variants and haematological indices have not yet been investigated in African SCD patients. This study aims to determine the associations between these variants and haematological indices in SCD patients from Cameroon and Senegal compared to non-SCD individuals. Aim: Investigating the association of 13 novel variants previously found in the Trans-Omics for Precision Medicine (TOPMed) project to haematological indices in African SCD patients. Methods: The 14 variants from the TOPMed study will be genotyped using a TaqMan Assay platform. Sanger sequencing will then be performed on 10% of the cohort to validate the TaqMan Assay result. The application R will be used for the descriptive statistics using sociodemographic, clinical, and haematological indices to evaluate the cases and controls. The Hardy-Weinberg Equilibrium will be used to ensure quality control. A linear regression analysis will be performed in R to determine the association between the variants and RBC phenotypes. Expected results: We are expecting to find an association between the SNPs and haematologocial indices. Conclusion: The variants of focus in this study were prioritized using WGS data, therefore capturing variants that are usually not included in GWAS arrays or imputation reference panels. The associations of the 13 novel variants with haematological indices have not yet been tested in African SCD populations. The results of this investigation could positively impact future studies into predictive modelling using haematological indices.

5613260 A PHASE -- IIA-IIB, OPEN LABEL, SINGLE CENTER TRIAL TO STUDY SAFETY, TOLERABILITY AND EFFICACY OF MEMANTINE AS SUPPORTIVE LONG-TERM TREATMENT OF SICKLE CELL PATIENTS: TRIAL DESIGN AND ENROLLMENT

Background: Sickle cell disease (SCD) is a life-threatening hematological disorder with a high rate of long-term comorbidities and a significant socioeconomic burden. Anemia, vaso-occlusive and pain crises, infections, and a high rate of long-term comorbidities are hallmarks of SCD. The pathophysiology of SCD is due to a single point mutation (Glu6Val) that causes polymerization of the mutant hemoglobin (HbS), resulting in red blood cells (RBC) sickling. Inflammation, hemolysis, microvascular obstruction, and organ damage characterize the clinical expression of SCD. The standard of care includes Hydroxycarbamide, symptomatic treatment, blood transfusions, new drugs, and bone marrow transplant as curative therapy. Our recent findings showed that N-methyl D-aspartate receptors (NMDARs) are up-regulated SCD-RBCs and Ca2+ uptake via NMDARs affects SCD-RBC hydration, and hypoxia-induced sickling (Hanggi P. et al., 2014). Inhibition of NMDARs with Memantine, a drug used in Alzheimer’s disease, emerges as a novel treatment in SCD. Memantine administration in 6 SCD adults demonstrated safety (Hegemann I. et al., 2020); and suggested a reduction of RBCs irreversible sickling (Makhro A, et al., 2020). Aims: This phase IIa-IIb open-label, single-center trial aims to study the safety, tolerability, and efficacy of Memantine in adults and teenagers with SCD (NCT03247218). Methods: This trial enrolled 23 SCD (SS and Sβ Thalassemia) 17 patients received Memantine for at least one year period, Hydroxycarbamide was continued administered. Starting dose of Memantine was 5 mg/day with escalation every 3 months, up to 15 mg/day in teenagers and up to 20 mg/day for adults. Patients were followed up monthly, a total of 6762 patients/days of follow-up were summarized. Results: The only safety events reported were transient headache and constipation in two patients; no adverse side effects were noticed. Overall, the number of admission days and emergency room visits decreased compared to the pre-screen period. The hemoglobin and fetal hemoglobin levels remain stable despite less use of blood transfusions; based on preliminary laboratory analysis a trend of reduction in hemolytic and inflammatory parameters can be noticed. Conclusions: The results of this phase IIa-IIb trial suggested safety and probable clinical benefit in long-term treatment with Memantine in SCD adolescents and adults. Full results of this trial are anticipated at the end of 2022 and a phase III trial will be required for the final assessment of the clinical benefit of Memantine treatment in SCD.

5613417 CO-TREATMENT OF ERYTHROID CELLS ISOLATED FROM Β039-THALASSEMIA PATIENTS WITH CRISPR-CAS9 GENE EDITING AND FETAL HEMOGLOBIN INDUCTION

Background: Gene editing (GE) can be considered among the most promising strategies to correct hereditary mutations in a variety of monogenetic diseases, including β-thalassemia. We have elsewhere reported that CRISPR-Cas9 gene editing can be proposed for efficient correction of the β039-thalassemia mutation. GE is suitable for personalized approaches in precision medicine for β-thalassemia [1]. Aims: On the other hand, robust evidence demonstrate that increased production of fetal hemoglobin (HbF) can be beneficial for β-thalassemia patients. This can be obtained also using CRISPR-Cas9 based protocols. In this case, the objective of the gene editing strategy is (a) the silencing of transcriptional repressors of the γ-globin genes (such as BCL11A), (b) the disruption of their regulatory binding sites within the γ-globin genes, or (c) the induction of natural hereditary persistence of fetal hemoglobin mutations in the γ-globin gene or the b-like-globin gene cluster. Our study aimed to verify whether the de novo production of adult hemoglobin (HbA) using CRISPR-Cas9 gene editing can be combined with HbF induction protocols based on the use of repositioned drugs. Results: Gene Editing of the β039-thalassemia mutation was obtained by the CRISPR-Cas9 method; gene correction and transcription of the corrected gene have been analyzed by allele-specific droplet digital PCR and RT-qPCR, respectively; production of HbA and HbF was studied by HPLC and Western blotting. For HbF induction, the repurposed drug sirolimus (rapamycin) was used. The data obtained conclusively demonstrate that maximal production of HbA and HbF is obtained in GE-corrected, sirolimus-induced erythroid progenitors from β039-thalassemia patients. In conclusion, GE and HbF in-duction might be used in combination in order to achieve the de novo production of HbA together with increased production of induced HbF. Acknowledgements This study was sustained by the Wellcome Trust (innovator award 208872/Z/17/Z) and by AIFA (AIFA-2016-02364887). The research leading to these results has received funding also from the UE THALAMOSS Project (Thalassemia Modular Stratification System for Personalized Therapy of Βeta-Thalassemia; no. 306201-FP7-HEALTH-2012-INNOVATION-1). We thanks A.L.T. (Associazione per la lotta alla Talassemia) “Rino Vullo” - Ferrara, and A.V.L.T. (Associazione Veneta per la Lotta alla Talassemia) “Elio Zago” - APS – Rovigo and Association “Tutti per Chiara”, Montagnana, PD, Italy. Reference 1. Cosenza L.C., Gasparello J., Romanini N., Zurlo M., Zuccato C., Gambari R., Finotti A. Efficient CRISPR-Cas9-based genome editing of β-globin gene on erythroid cells from homozygous β039-thalassemia patients. Mol Ther Methods Clin Dev 2021;21:507−523. https://doi.org/10.1016/j.omtm.2021.03.025

Design, Synthesis, and Evaluation of Allosteric Effectors for Hemoglobin.

ConspectusSickle cell disease (SCD) is an inherited blood disorder caused by a point mutation in hemoglobin (Hb), the protein in the red blood cell (RBC) responsible for the transport of oxygen (O2) throughout the body. The mutation leads to the expression of sickle cell hemoglobin (HbS). Both Hb and HbS exist in equilibrium between oxygenated and deoxygenated forms; however, deoxygenated HbS can polymerize to form long fibers which distort the shape of RBCs into the characteristic sickled shape. The misshapen RBCs can obstruct blood vessels and capillaries, resulting in a vaso-occlusive crisis. Vaso-occulsion deprives tissues and organs of O2 and can cause intense pain which often results in hospitalization. Chronic organ damage is a major cause of reduced life expectancy for SCD patients.Allosteric effectors are molecules which regulate protein function. HbS allosteric effectors can be used to decrease polymerization by stabilizing the oxygenated form of HbS, which leads to an increase in O2 uptake and a decrease in the sickling of RBCs. Allosteric effectors that have been evaluated for the treatment of SCD include vanillin, 5-hydroxymethyl furfural (5-HMF), and voxelotor, which was approved by the U.S. Food and Drug Administration (FDA) for the treatment of SCD in 2019. 5-HMF did not progress to phase III clinical trials since it suffered from rapid metabolic degradation. However, several derivatives of 5-HMF and vanillin have been synthesized and evaluated as potential candidates for SCD treatment. Derivatives of these compounds have shown promise, but their shortcomings, such as high levels of oxidative metabolism, have prevented them from progressing into marketable drugs. Our efforts have produced multiple 5-HMF derivatives which have been evaluated for their potential to treat SCD. Each derivative was evaluated for its ability to increase O2 affinity (i.e., P50, the partial pressure at which hemoglobin is 50% saturated with O2). The synthesized aryl ether derivatives were evaluated, and results suggest that compounds with multiple aromatic aldehydes may have enhanced biological properties. One such derivative, compound 5, which features two furan aldehyde rings, exhibited increased O2 affinity (P50 = 8.82 ± 1.87 mmHg) over that of unmodified Hb (P50 = 13.67 ± 0.22 mmHg). Future studies include obtaining crystal structures of the 5-HMF derivatives complexed with HbS to confirm the protein-allosteric effector interactions.

Structural and oxidative investigation of a recombinant high-yielding fetal hemoglobin mutant.

Human fetal hemoglobin (HbF) is an attractive starting protein for developing an effective agent for oxygen therapeutics applications. This requires that HbF can be produced in heterologous systems at high levels and in a homogeneous form. The introduction of negative charges on the surface of the α-chain in HbF can enhance the recombinant production yield of a functional protein in Escherichia coli. In this study, we characterized the structural, biophysical, and biological properties of an HbF mutant carrying four additional negative charges on each α-chain (rHbFα4). The 3D structure of the rHbFα4 mutant was solved with X-ray crystallography at 1.6Å resolution. Apart from enabling a higher yield in recombinant protein production in E. coli, we observed that the normal DNA cleavage activity of the HbF was significantly lowered, with a four-time reduced rate constant for the rHbFα4 mutant. The oxygen-binding properties of the rHbFα4 mutant were identical to the wild-type protein. No significant difference between the wild-type and rHbFα4 was observed for the investigated oxidation rates (autoxidation and H2O2-mediated ferryl formation). However, the ferryl reduction reaction indicated some differences, which appear to be related to the reaction rates linked to the α-chain.

Magnetic Fractionation of Red Blood Cells Based on Oxygen Saturation in Sickle Cell Disease

Anemias are a broad classification of red blood cell (RBC) disorders which effectively result in inadequate oxygen transport in the body. Sickle Cell Disease (SCD) results from a point mutation in hemoglobin, and the hemoglobin-oxygen equilibrium becomes right shifted (i.e., SCD RBCs do not oxygen saturate to the same degree as healthy RBCs). RBC exchange transfusion is a common clinical practice to increase the oxygen carrying capacity of a SCD patient - a process by which the patient's own RBCs are exchanged with healthy donor blood. During this process, large amounts of donor RBCs are discarded along with the patients SCD RBCs. Here we present a new RBC separation modality based on hemoglobin-oxygen equilibrium.It is well known that RBCs become magnetized as a function of the oxygen saturation of its hemoglobin. Due to the difference in hemoglobin-oxygen equilibrium for healthy and SCD RBCs we demonstrate a magnetic method of fractionating RBCs based on oxygen saturation. In our study, we use the SCD transfusion waste product and sort this sample into healthy fractions and SCD fractions using a field flow permeant magnetic separator. Under variable oxygen tension of the sample, we preformed the magnetic sorting.By incorporation of magnetic separation into RBC exchange processes, more efficient selection, and removal of SCD RBCs can be accomplished. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Modified and Novel Lorrca Oxygenscan Measurement Parameters with Physiological Relevance

BACKROUND: Despite arising from a single-point hemoglobin (Hb) mutation (βGlu6 replaced by valine), patients with sickle cell disease (SCD) display wide phenotype variation. This likely arises from sickling and its resulting pathology having multiple physiologic triggers, many of which are red blood cell (RBC) related, i.e., sickle hemoglobin (HbS) and fetal hemoglobin (HbF) concentration, membrane permeability, cell hydration, etc. These triggers are inadequately captured by single measurement parameters and require a more holistic functional RBC assessment (reflecting their composite effects). To provide such, oxygen (O2) gradient ektacytometry (LORRCA oxygenscan RR Mechatronics) was designed as a commercial, rapid, reproducible, and automated laboratory assay, which mimics in vivo physiology, quantifying RBC deformability across the physiological O2 gradient. This assay captures the process of sickling/unsickling of a RBC population when challenged with deoxygenation and reoxygenation. Significant variance in the oxygenscan point of sickling measurement was observed (POS - defined as the pO2at which EImax declined 5% - representing the initiation of sickling), despite following all guidelines for sample handling. EI fluctuation at higher O2 partial pressure (pO2) was noted as the cause for this. We consequently modified the POS determination, centering our analysis on EI rate of change (i.e., the first derivative of the EI trace), rather than the single point software determination. We further identified two new analyses parameters which reflect aspects of RBC physiology of potential physiologic benefit in SCD. Firstly, we used the rate of change in EI (a first derivative-based analysis) on the reoxygenation arm of the LORRCA trace, to identify the pO2 at which EI was observed to significantly increase from EImin, defining this as the point of unsickling (POU). Lastly, we set about quantifying the hysteresis between the O2 unloading and O2 loading EI curves, calling this the area between curves. METHODS: RBCs from SCD patients were incubated with the Hb allosteric effector 5-hydroxymethylfurfural (5HMF; 20% Hct, 37C, HMF: 0-5mM, 0-3 hrs); 5HMF binds to Hb NH2 terminal αValine1, increasing oxyHbS abundance, thereby inhibiting the mechanistic trigger for deoxygenation induced HbS polymerization.Oxygenscans were performed with elongation index (EI) determined by laser light diffraction, across the physiologic O2 gradient (pO2 ~150-10 mmHg; sample gassed with nitrogen ~1300 seconds followed by swift re-oxygenation in room air ~280 seconds). Samples were analyzed utilizing standard parameters offered by the LORRCA software in addition to our modified POS determination and analysis of POU and area between curves. RESULTS: Little change in mean POS was observed in baseline measurements (minus 5HMF) between the standard LORRCA software analysis and our modified analysis (POS pO2 = 55.1 ± 20.4 vs 56.9 ± 10.1mmHg mean±SD, respectively), however the coefficient of variance (COV) of our modified analysis was ~20% lower. The same trend was observed in samples across all doses of 5HMF. No significant difference in POS was detected with the standard analysis between any of the 5HMF doses. However, a significant reduction in POS between 0 and 5mM 5HMF final was determined with our modified analysis (POS pO2 = 56.9 ± 10.1 vs 39.8 ±11.6mmHg, respectively). A significant difference was also observed in the POU between 0 and 5mM HMF (POU pO2 = 38.3 ± 9.1 vs 21.3 ± 5.7mmHg, respectively) with HMF resulting in a lower pO2 at which unsickling was observed. This has obvious patient benefit implications, with SRBCs regaining deformability at lower pO2. Finally, a significant difference was also observed in the area between the deoxygenation and reoxygenation EI curves between 0 and 5mM HMF (area between curve = 8.9 ± 4.6 vs 4.6 ± 1.6AU, respectively). This smaller area between curves implies less change in deformability behavior between oxygenation states in the presence of 5HMF. SUMMARY: We have developed a more robust analysis methodology for the determination of POS by oxygenscan. This appears to work well even on sub-optimal scans and can detect a change in sickling characteristics for the allosteric modifier 5HMF. In addition, we suggest two further analyses parameters for oxygenscan (POU and area between curves) which reflect aspects of RBC physiology of potential physiologic benefit in SCD.