Development and IND-enabling studies of a novel Cas9 genome-edited autologous CD34+ cell therapy to induce fetal hemoglobin for sickle cell disease.

Sickle cell disease (SCD) is a common, severe genetic blood disorder. Current pharmacotherapies are partially effective and allogeneic hematopoietic stem cell transplantation is associated with immune toxicities. Genome editing of patient hematopoietic stem cells (HSCs) to reactivate fetal hemoglobin (HbF) in erythroid progeny offers an alternative potentially curative approach to treat SCD. Although the FDA released guidelines for evaluating genome editing risks, it remains unclear how best to approach pre-clinical assessment of genome-edited cell products. Here, we describe rigorous pre-clinical development of a therapeutic γ-globin gene promoter editing strategy that supported an investigational new drug application cleared by the FDA. We compared γ-globin promoter and BCL11A enhancer targets, identified a potent HbF-inducing lead candidate, and tested our approach in mobilized CD34+ hematopoietic stem progenitor cells (HSPCs) from SCD patients. We observed efficient editing, HbF induction to predicted therapeutic levels, and reduced sickling. With single-cell analyses, we defined the heterogeneity of HbF induction and HBG1/HBG2 transcription. With CHANGE-seq for sensitive and unbiased off-target discovery followed by targeted sequencing, we did not detect off-target activity in edited HSPCs. Our study provides a blueprint for translating new ex vivo HSC genome editing strategies toward clinical trials for treating SCD and other blood disorders.

Transient neonatal hemolytic anemia due to the novel gamma globin gene mutation HBG2:C.290T>C, p.Leu97Pro (hemoglobin Wareham).

Unstable gamma globin variants can cause transient neonatal hemolytic anemia. We have identified a novel variant in a newborn who presented with jaundice and anemia requiring phototherapy and red blood cell transfusion. The patient was found to be heterozygous for the mutation HGB2:c.290T>C, p.Leu97Pro, which we have termed hemoglobin (Hb) Wareham. This substitution is expected to generate an unstable hemoglobin with increased oxygen affinity based on the homologous mutation previously described in the beta globin gene, which is termed as Hb Debrousse. The patient fully recovered by 9 months of age as expected with the transition from fetal to adult hemoglobin.

Synthesis and evaluation of resveratrol derivatives as fetal hemoglobin inducers.

Resveratrol (RVT) derivatives (10a-i) were designed, synthesized, and evaluated for their potential as gamma-globin inducers in treating Sickle Cell Disease (SCD) symptoms. All compounds were able to release NO at different levels ranging from 0 to 26.3%, while RVT did not demonstrate this effect. In vivo, the antinociceptive effect was characterized using an acetic acid-induced abdominal contortion model. All compounds exhibited different levels of protection, ranging from 5.9 to 37.3%; the compound 10a was the most potent among the series. At concentrations between 3.13 and 12.5 µM, the derivative 10a resulted in a reduction of 41.1-64.3% in the TNF-α levels in the supernatants of macrophages that were previously LPS-stimulated. This inhibitory effect was higher than that of RVT used as the control. In addition, the compound 10a and RVT induced double the production of the gamma-globin chains (γG + Î³A), compared to the vehicle, using CD34+ cells. Compound 10a also did not induce membrane perturbation and it was not mutagenic in the in vivo assay. Thus, compound 10a emerged as a new prototype of the gamma-globin-inducer group with additional analgesic and anti-inflammatory activities and proving to be a useful alternative to treat SCD symptoms.

Tough decoy targeting of predominant let-7 miRNA species in adult human hematopoietic cells.

BACKGROUND: In humans, the heterochronic cascade composed of the RNA-binding protein LIN28 and its major target, the let-7 family of microRNAs (miRNAs), is highly regulated during human erythroid ontogeny. Additionally, down-regulation of the let-7 miRNAs in cultured adult CD34(+) cells or the over-expression of LIN28 in cultured erythrocytes from pediatric patients with HbSS genotype causes increased levels of fetal hemoglobin (HbF) in the range of 19-40% of the total. Therefore, we hypothesized that focused targeting of individual let-7 miRNA family members would exhibit regulatory effect on HbF expression in human adult erythroblasts. METHODS: The expression levels of mature let-7 family members were measured by RT-qPCR in purified cell populations sorted from peripheral blood. To study the effects of let-7 miRNAs upon globin expression, a lentiviral construct that incorporated the tough decoy (TuD) design to target let-7a or let-7b was compared with empty vector controls. Transductions were performed in CD34(+) cells from adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Downstream analyses included RT-qPCR, Western blot and HPLC for the characterization of adult and fetal hemoglobins. RESULTS: The expression of individual let-7 miRNA family members in adult peripheral blood cell populations demonstrated that let-7a and let-7b miRNAs are expressed at much higher levels than the other let-7 family members in purified adult human blood cell subsets with expression being predominantly in reticulocytes. Therefore, we focused this study upon the targeted inhibition of let-7a and let-7b with the TuD design to explore its effects upon developmentally-timed erythroid genes. Let-7a-TuD transductions significantly increased gamma-globin mRNA expression and HbF to an average of 38%. Let-7a-TuD also significantly decreased the mRNA expression of some ontogeny-regulated erythroid genes, namely CA1 and GCNT2. In addition, the erythroid-related transcription factors BCL11A and HMGA2 were down- and up-regulated, respectively, by let-7a-TuD, while ZBTB7A, KLF1 and SOX6 remained unchanged. CONCLUSIONS: Overall, our data demonstrate that let-7 miRNAs are differentially expressed in human hematopoietic cells, and that targeted inhibition of the highly-expressed species of this family is sufficient for developmentally-specific changes in gamma-globin expression and HbF levels.

Reactivation of Fetal Hemoglobin for Treating ß-Thalassemia and Sickle Cell Disease.

Reactivation of fetal hemoglobin (HbF) in adult hematopoietic cells has the potential for great clinical benefit in patients bearing deleterious mutations in the ß-globin gene, such as ß-thalassemia and sickle cell disease (SCD), since increasing the production of HbF can compensate for underproduction of ß-globin chains (in ß-thalassemia) and it can also disrupt sickle hemoglobin polymerization (in SCD). Thus for the past few decades, concerted efforts have been made to identify an effective way to induce the synthesis of HbF in adult erythroid cells for potential therapeutic relief from the effects of these ß-globinopathies. Chemical inducers of HbF as well as a number of transcription factors that are able to reactivate HbF synthesis in vitro and in vivo in adult erythroid cells have been identified. However, there has been only limited success in attempts to manipulate either the drugs or regulatory proteins, and in only a fraction of patients, and there is wide variation in individual response to these drugs or transcription factors. These studies highlight the importance for understanding the molecular mechanisms underlying hemoglobin switching so that future studies can be designed to treat these disorders.

Comparison of different methods for erythroid differentiation in the K562 cell line.

OBJECTIVE: To compare methods for erythroid differentiation of K562 cells that will be promising in the treatment of beta-thalassemia by inducing γ-globin synthesis. RESULTS: Cells were treated separately with: RPMI 1640 medium without glutamine, RPMI 1640 medium without glutamine supplemented with 1 mM sodium butyrate, RPMI 1640 medium supplemented with 1 mM sodium butyrate, 25 µg cisplatin/ml, 0.1 µg cytosine arabinoside/ml. The highest differentiation (84 %) with minimum toxicity was obtained with cisplatin at 15 µg /ml. Real-time RT-PCR showed that expression of the γ-globin gene was significantly higher in the cells differentiated with cisplatin compared to undifferentiated cells (P < 0.001). CONCLUSIONS: Cisplatin is useful in the experimental therapy of ß-globin gene defects and can be considered for examining the basic mechanism of γ-reactivation.

Transcriptional regulators of fetal hemoglobin.

Sickle cell anemia is a hereditary disease caused by sickle-shaped red blood cells that can lead to vaso-occlusive crises. Treatment options are currently limited, highlighting the need to develop new clinical approaches. Studies demonstrated that elevated levels of fetal hemoglobin (Hb F) are associated with a reduction of mortality and morbidity in sickle cell anemia patients. In light of this, researchers have been trying to elucidate the transcriptional regulation of Hb F to develop new therapeutic interventions. The present study aimed to present the main transcription factors of Hb F and discuss the clinical feasibility of these molecular targets. Two search strategies were used in the PubMed, SciELO, and LILACS databases between July and August 2023 to conduct this review. Manual searches were also conducted by checking references of potentially eligible studies. Eligibility criteria consisted of clinical trials and cohort studies from the last five years that investigated transcription factors associated with Hb F. The transcription factors investigated in at least four eligible studies were included in this review. As a result, 56 eligible studies provided data on the BCL11A, LRF, NF-Y, GATA1, KLF1, HRI, ATF4, and MYB factors. The studies demonstrated that Hb F is cooperatively regulated by transcription factors with the BCL11A factor appearing to be the most specific target gene for γ-globin induction. Although these data are promising, there are still significant gaps and intervention limitations due to the adverse functions of the target genes. New studies that clarify the aspects and functionalities of Hb F regulators may enable new clinical approaches for sickle cell anemia patients.

Keap1-Nrf2 Heterodimer: A Therapeutic Target to Ameliorate Sickle Cell Disease.

Sickle cell disease (SCD) is a monogenic inheritable disease characterized by severe anemia, increased hemolysis, and recurrent, painful vaso-occlusive crises due to the polymerization of hemoglobin S (HbS)-generated oxidative stress. Up until now, only four drugs are approved for SCD in the US. However, each of these drugs affects only a limited array of SCD pathologies. Importantly, curative therapies, such as gene therapy, or hematopoietic stem cell transplantation are not available for every patient because of their high costs, availability of donor matching, and their serious adverse effects. Therefore, there is an unmet medical need for novel therapeutic strategies that target broader SCD sequelae. SCD phenotypic severity can be alleviated by increasing fetal hemoglobin (HbF) expression. This results in the inhibition of HbS polymerization and thus sickling, and a reduction in oxidative stress. The efficacy of HbF is due to its ability to dilute HbS levels below the threshold required for polymerization and to influence HbS polymer stability in RBCs. Nuclear factor-E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein-1 (Keap1)-complex signaling is one of the most important cytoprotective signaling controlling oxidative stress. Nrf2 is present in most organs and, after dissociation from Keap1, it accumulates in the cytoplasm, then translocates to the nucleus where it binds to the antioxidant response element (ARE) sequences and increases the expression of various cytoprotective antioxidant genes. Keeping this in mind, various researchers have proposed a role of multiple agents, more importantly tert-Butylhydroquinone (tBHQ), curcumin, etc., (having electrophilic properties) in inhibiting keap1 activity, so that Nrf2 can translocate to the nucleus to activate the gamma globin gene, thus maintaining alpha-hemoglobin-stabilizing protein (AHSP) and HbF levels. This leads to reduced oxidative stress, consequently minimizing SCD-associated complications. In this review, we will discuss the role of the Keap-1-Nrf2 complex in hemoglobinopathies, especially in SCD, and how this complex might represent a better target for more effective treatment options.

The BACH1 inhibitor ASP8731 inhibits inflammation and vaso-occlusion and induces fetal hemoglobin in sickle cell disease.

In sickle cell disease (SCD), heme released during intravascular hemolysis promotes oxidative stress, inflammation, and vaso-occlusion. Conversely, free heme can also activate expression of antioxidant and globin genes. Heme binds to the transcription factor BACH1, which represses NRF2-mediated gene transcription. ASP8731, is a selective small molecule inhibitor of BACH1. We investigated the ability of ASP8731 to modulate pathways involved in SCD pathophysiology. In HepG2 liver cells, ASP8731 increased HMOX1 and FTH1 mRNA. In pulmonary endothelial cells, ASP8731 decreased VCAM1 mRNA in response to TNF-α and blocked a decrease in glutathione in response to hemin. Townes-SS mice were gavaged once per day for 4 weeks with ASP8731, hydroxyurea (HU) or vehicle. Both ASP8731 and HU inhibited heme-mediated microvascular stasis and in combination, ASP8731 significantly reduced microvascular stasis compared to HU alone. In Townes-SS mice, ASP8731 and HU markedly increased heme oxygenase-1 and decreased hepatic ICAM-1, NF-kB phospho-p65 protein expression in the liver, and white blood cell counts. In addition, ASP8731 increased gamma-globin expression and HbF+ cells (F-cells) as compared to vehicle-treated mice. In human erythroid differentiated CD34+ cells, ASP8731 increased HGB mRNA and increased the percentage of F-cells 2-fold in manner similar to HU. ASP8731 and HU when given together induced more HbF+ cells compared to either drug alone. In CD34+ cells from one donor that was non-responsive to HU, ASP8731 induced HbF+ cells ~2-fold. ASP8731 and HU also increased HBG and HBA, but not HBB mRNA in erythroid differentiated CD34+ cells derived from SCD patients. These data indicate that BACH1 may offer a new therapeutic target to treat SCD.

Bioinformatic Tools for the Identification of MicroRNAs Regulating the Transcription Factors in Patients with ß-Thalassemia.

ß-thalassemia is a significant health issue worldwide, with approximately 7% of the world's population having defective hemoglobin genes. MicroRNAs (miRNAs) are short noncoding RNAs regulating gene expression at the post-transcriptional level by targeting multiple gene transcripts. The levels of fetal hemoglobin (HbF) can be increased by regulating the expression of the γ-globin gene using the suppressive effects of miRNAs on several transcription factors such as MYB, BCL11A, GATA1, and KLF. An early step in discovering miRNA:mRNA target interactions is the computational prediction of miRNA targets that can be later validated with wet-lab investigations. This review highlights some commonly employed computational tools such as miRBase, Target scan, DIANA-microT-CDS, miRwalk, miRDB, and micro-TarBase that can be used to predict miRNA targets. Upon comparing the miRNA target prediction tools, 4 main aspects of the miRNA:mRNA target interaction are shown to include a few common features on which most target prediction is based: conservation sites, seed match, free energy, and site accessibility. Understanding these prediction tools' usage will help users select the appropriate tool and interpret the results accurately. This review will, therefore, be helpful to peers to quickly choose a list of the best miRNAs associated with HbF induction. Researchers will obtain significant results using these bioinformatics tools to establish a new important concept in managing ß-thalassemia and delivering therapeutic strategies for improving their quality of life.

Safe and efficient in vivo hematopoietic stem cell transduction in nonhuman primates using HDAd5/35++ vectors.

We tested a new in vivo hematopoietic stem cell (HSC) transduction/selection approach in rhesus macaques using HSC-tropic, integrating, helper-dependent adenovirus vectors (HDAd5/35++) designed for the expression of human γ-globin in red blood cells (RBCs) to treat hemoglobinopathies. We show that HDAd5/35++ vectors preferentially transduce HSCs in vivo after intravenous injection into granulocyte colony-stimulating factor (G-CSF)/AMD3100-mobilized animals and that transduced cells return to the bone marrow and spleen. The approach was well tolerated, and the activation of proinflammatory cytokines that are usually associated with intravenous adenovirus vector injection was successfully blunted by pre-treatment with dexamethasone in combination with interleukin (IL)-1 and IL-6 receptor blockers. Using our MGMTP140K-based in vivo selection approach, γ-globin+ RBCs increased in all animals with levels up to 90%. After selection, the percentage of γ-globin+ RBCs declined, most likely due to an immune response against human transgene products. Our biodistribution data indicate that γ-globin+ RBCs in the periphery were mostly derived from mobilized HSCs that homed to the spleen. Integration site analysis revealed a polyclonal pattern and no genotoxicity related to transgene integrations. This is the first proof-of-concept study in nonhuman primates to show that in vivo HSC gene therapy could be feasible in humans without the need for high-dose chemotherapy conditioning and HSC transplantation.

Targeting fetal hemoglobin expression to treat ß hemoglobinopathies.

INTRODUCTION: Sickle cell disease and ß thalassemia are the principal ß hemoglobinopathies. The complex pathophysiology of sickle cell disease is initiated by sickle hemoglobin polymerization. In ß thalassemia, insufficient ß-globin synthesis results in excessive free α globin, ineffective erythropoiesis, and severe anemia. Fetal hemoglobin (HbF) prevents sickle hemoglobin polymerization; in ß thalassemia HbF compensates for the deficit of normal hemoglobin. When HbF constitutes about a third of total cell hemoglobin, the complications of sickle cell disease are nearly totally prevented. Similarly, sufficient HbF in ß thalassemia diminishes or prevents ineffective erythropoiesis and hemolysis. AREAS COVERED: This article examines the pathophysiology of ß hemoglobinopathies, the physiology of HbF, intracellular distribution, and the regulation of HbF expression. Inducing high levels of HbF by targeting its regulatory pathways pharmacologically or with cell-based therapeutics provides major clinical benefit and perhaps a 'cure.' EXPERT OPINION: Erythrocytes must contain about 10 pg of HbF to 'cure' sickle cell disease. If HbF is the only hemoglobin present, much higher levels are needed to 'cure' ß thalassemia. These levels of HbF can be obtained by different iterations of gene therapy. Small molecule drugs that can achieve even modest pancellular HbF concentrations are a major unmet need.

A Small Key for a Heavy Door: Genetic Therapies for the Treatment of Hemoglobinopathies.

Throughout the past decades, the search for a treatment for severe hemoglobinopathies has gained increased interest within the scientific community. The discovery that ɤ-globin expression from intact HBG alleles complements defective HBB alleles underlying ß-thalassemia and sickle cell disease, has provided a promising opening for research directed at relieving ɤ-globin repression mechanisms and, thereby, improve clinical outcomes for patients. Various gene editing strategies aim to reverse the fetal-to-adult hemoglobin switch to up-regulate ɤ-globin expression through disabling either HBG repressor genes or repressor binding sites in the HBG promoter regions. In addition to these HBB mutation-independent strategies involving fetal hemoglobin (HbF) synthesis de-repression, the expanding genome editing toolkit is providing increased accuracy to HBB mutation-specific strategies encompassing adult hemoglobin (HbA) restoration for a personalized treatment of hemoglobinopathies. Moreover, besides genome editing, more conventional gene addition strategies continue under investigation to restore HbA expression. Together, this research makes hemoglobinopathies a fertile ground for testing various innovative genetic therapies with high translational potential. Indeed, the progressive understanding of the molecular clockwork underlying the hemoglobin switch together with the ongoing optimization of genome editing tools heightens the prospect for the development of effective and safe treatments for hemoglobinopathies. In this context, clinical genetics plays an equally crucial role by shedding light on the complexity of the disease and the role of ameliorating genetic modifiers. Here, we cover the most recent insights on the molecular mechanisms underlying hemoglobin biology and hemoglobinopathies while providing an overview of state-of-the-art gene editing platforms. Additionally, current genetic therapies under development, are equally discussed.

Fetal Hemoglobin in Sickle Hemoglobinopathies: High HbF Genotypes and Phenotypes.

Fetal hemoglobin (HbF) usually consists of 4 to 10% of total hemoglobin in adults of African descent with sickle cell anemia. Rarely, their HbF levels reach more than 30%. High HbF levels are sometimes a result of ß-globin gene deletions or point mutations in the promoters of the HbF genes. Collectively, the phenotype caused by these mutations is called hereditary persistence of fetal hemoglobin, or HPFH. The pancellularity of HbF associated with these mutations inhibits sickle hemoglobin polymerization in most sickle erythrocytes so that these patients usually have inconsequential hemolysis and few, if any, vasoocclusive complications. Unusually high HbF can also be associated with variants of the major repressors of the HbF genes, BCL11A and MYB. Perhaps most often, we lack an explanation for very high HbF levels in sickle cell anemia.

Integrating HDAd5/35++ Vectors as a New Platform for HSC Gene Therapy of Hemoglobinopathies.

We generated an integrating, CD46-targeted, helper-dependent adenovirus HDAd5/35++ vector system for hematopoietic stem cell (HSC) gene therapy. The ∼12-kb transgene cassette included a ß-globin locus control region (LCR)/promoter driven human γ-globin gene and an elongation factor alpha-1 (EF1α)-mgmtP140K expression cassette, which allows for drug-controlled increase of γ-globin-expressing erythrocytes. We transduced bone marrow lineage-depleted cells from human CD46-transgenic mice and transplanted them into lethally irradiated recipients. The percentage of γ-globin-positive cells in peripheral blood erythrocytes in primary and secondary transplant recipients was stable and greater than 90%. The γ-globin level was 10%-20% of adult mouse globin. Transgene integration, mediated by a hyperactive Sleeping Beauty SB100x transposase, was random, without a preference for genes. A second set of studies was performed with peripheral blood CD34+ cells from mobilized donors. 10 weeks after transplantation of transduced cells, human cells were harvested from the bone marrow and differentiated ex vivo into erythroid cells. Erythroid cells expressed γ-globin at a level of 20% of adult α-globin. Our studies suggest that HDAd35++ vectors allow for efficient transduction of long-term repopulating HSCs and high-level, almost pancellular γ-globin expression in erythrocytes. Furthermore, our HDAd5/35++ vectors have a larger insert capacity and a safer integration pattern than currently used lentivirus vectors.

Original Research: Stable expression of miR-34a mediates fetal hemoglobin induction in K562 cells.

Sickle cell anemia is a common genetic disorder caused by a point mutation in the sixth codon of the ß-globin gene affecting people of African descent worldwide. A wide variety of clinical phenotypes ranging from mild to severe symptoms and complications occur due to hemoglobin S polymerization, red blood cell sickling, and vaso-occlusion. Research efforts are ongoing to develop strategies of fetal hemoglobin (HbF; α2γ2) induction to inhibit sickle hemoglobin polymerization and improve clinical outcomes. Insights have been gained from investigating mutations in the ß-globin locus or transcription factors involved in the mechanisms of hemoglobin switching. Recent efforts to expand molecular targets that modulate γ-globin expression involve microRNAs that work through posttranscriptional gene regulation. Therefore, the goal of our study was to identify novel microRNA genes involved in fetal hemoglobin expression. Using in silico analysis, we identified a miR-34a binding site in the γ-globin mRNA which was tested for functional relevance. Stable expression of the shMIMIC miR-34a lentivirus vector increased fetal hemoglobin levels in single cell K562 clones consistent with silencing of a γ-globin gene repressor. Furthermore, miR-34a promoted cell differentiation supported by increased expression of KLF1, glycophorin A, and the erythropoietin receptor. Western blot analysis of known negative regulators of γ-globin including YY1, histone deacetylase 1, and STAT3, which are regulated by miR-34a showed no change in YY1 and histone deacetylase 1 levels; however, total- and phosphorylated-STAT3 levels were decreased in single cell miR-34a K562 clones. These data support a mechanism of fetal hemoglobin activation by miR-34a involving STAT3 gene silencing.

Current and future alternative therapies for beta-thalassemia major.

Beta-thalassemia is a group of frequent genetic disorders resulting in the synthesis of little or no ß-globin chains. Novel approaches are being developed to correct the resulting α/ß-globin chain imbalance, in an effort to move beyond the palliative management of this disease and the complications of its treatment (e.g. life-long red blood cell transfusion, iron chelation, splenectomy), which impose high costs on healthcare systems. Three approaches are envisaged: fetal globin gene reactivation by pharmacological compounds injected into patients throughout their lives, allogeneic hematopoietic stem cell transplantation (HSCT), and gene therapy. HSCT is currently the only treatment shown to provide an effective, definitive cure for ß-thalassemia. However, this procedure remains risky and histocompatible donors are identified for only a small fraction of patients. New pharmacological compounds are being tested, but none has yet made it into common clinical practice for the treatment of beta-thalassemia major. Gene therapy is in the experimental phase. It is emerging as a powerful approach without the immunological complications of HSCT, but with other possible drawbacks. Rapid progress is being made in this field, and long-term efficacy and safety studies are underway.

Cis-vaccenic acid induces differentiation and up-regulates gamma globin synthesis in K562, JK1 and transgenic mice erythroid progenitor stem cells.

Gamma globin induction remains a promising pharmacological therapeutic treatment mode for sickle cell anemia and beta thalassemia, however Hydroxyurea remains the only FDA approved drug which works via this mechanism. In this regard, we assayed the γ-globin inducing capacity of Cis-vaccenic acid (CVA). CVA induced differentiation of K562, JK1 and transgenic mice primary bone marrow hematopoietic progenitor stem cells. CVA also significantly up-regulated γ-globin gene expression in JK-1 and transgenic mice bone marrow erythroid progenitor stem cells (TMbmEPSCs) but not K562 cells without altering cell viability. Increased γ-globin expression was accompanied by KLF1 suppression in CVA induced JK-1 cells. Erythropoietin induced differentiation of JK-1 cells 24h before CVA induction did not significantly alter CVA induced differentiation and γ-globin expression in JK-1 cells. Inhibition of JK-1 and Transgenic mice bone marrow erythroid progenitor stem cells Fatty acid elongase 5 (Elovl5) and Δ(9) desaturase suppressed the γ-globin inductive effects of CVA. CVA treatment failed to rescue γ-globin expression in Elovl5 and Δ(9)-desaturase inhibited cells 48 h post inhibition in JK-1 cells. The data suggests that CVA directly modulates differentiation of JK-1 and TMbmEPSCs, and indirectly modulates γ-globin gene expression in these cells. Our findings provide important clues for further evaluations of CVA as a potential fetal hemoglobin therapeutic inducer.

Sequence change in the HS2-LCR and Gg-globin gene promoter region of sickle cell anemia patients

The fetal hemoglobin (HbF) levels and betaS-globin gene haplotypes of 125 sickle cell anemia patients from Brazil were investigated. We sequenced the Gg- and Ag-globin gene promoters and the DNase I-2 hypersensitive sites in the locus control regions (HS2-LCR) of patients with HbF level disparities as compared to their ßS haplotypes. Sixty-four (51.2 percent) patients had CAR/Ben genotype; 36 (28.8 percent) Ben/Ben; 18 (14.4 percent) CAR/CAR; 2 (1.6 percent) CAR/Atypical; 2 (1.6 percent) Ben/Cam; 1 (0.8 percent) CAR/Cam; 1 (0.8 percent) CAR/Arab-Indian, and 1 (0.8 percent) Sen/Atypical. The HS2-LCR sequence analyses demonstrated a c.-10.677G>A change in patients with the Ben haplotype and high HbF levels. The Gg gene promoter sequence analyses showed a c.-157T>C substitution shared by all patients, and a c.-222_-225del related to the Cam haplotype. These results identify new polymorphisms in the HS2-LCR and Gg-globin gene promoter. Further studies are required to determine the correlation between HbF synthesis and the clinical profile of sickle cell anemia patients.