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The β-thalassemias are inherited genetic disorders affecting the hematopoietic system. In β-thalassemias, more than 350 mutations of the adult β-globin gene cause the low or absent production of adult hemoglobin (HbA). A clinical parameter affecting the physiology of erythroid cells is the excess of free α-globin. Possible experimental strategies for a reduction in excess free α-globin chains in β-thalassemia are CRISPR-Cas9-based genome editing of the β-globin gene, forcing "de novo" HbA production and fetal hemoglobin (HbF) induction. In addition, a reduction in excess free α-globin chains in β-thalassemia can be achieved by induction of the autophagic process. This process is regulated by the Unc-51-like kinase 1 (Ulk1) gene. The interplay with the PI3K/Akt/TOR pathway, with the activity of the α-globin stabilizing protein (AHSP) and the involvement of microRNAs in autophagy and Ulk1 gene expression, is presented and discussed in the context of identifying novel biomarkers and potential therapeutic targets for β-thalassemia. [ABSTRACT FROM AUTHOR]

Background/Objectives: in β-thalassemia, important clinical complications are caused by the presence of free α-globin chains in the erythroid cells of β-thalassemia patients. These free α-globin chains are present in excess as a result of the lack of β-globin chains to bind with; they tend to aggregate and precipitate, causing deleterious effects and overall cytotoxicity, maturation arrest of the erythroid cells and, ultimately, ineffective erythropoiesis. The chaperone protein α-hemoglobin-stabilizing protein (AHSP) reversibly binds with free α-globin; the resulting AHSP-αHb complex prevents aggregation and precipitation. Sirolimus (rapamycin) has been previously demonstrated to induce expression of fetal hemoglobin and decrease the excess of free α-globin chain in the erythroid cells of β-thalassemia patients. The objective of this study was to verify whether sirolimus is also able to upregulate AHSP expression in erythroid precursor cells (ErPCs) isolated from β-thalassemia patients. Methods: the expression of AHSP genes was analyzed by measuring the AHSP mRNA content by real-time quantitative PCR (RT-qPCR) and the AHSP protein production by Western blotting. Results: AHSP gene expression was found to be higher in ErPCs of β-thalassemia patients in comparison to ErPCs isolated from healthy subjects. In addition, AHSP expression was further induced by treatment of β-thalassemia ErPCs with sirolimus. Finally, AHSP mRNA was expressed at an increased level in ErPCs of sirolimus-treated β-thalassemia patients participating in the NCT03877809 Sirthalaclin clinical trial. Conclusions: this exploratory study suggests that AHSP expression should be considered as an endpoint in clinical trials based on sirolimus. [ABSTRACT FROM AUTHOR]

In this short review, we presented and discussed studies on the expression of globin genes in β-thalassemia, focusing on the impact of α-globin gene expression and α-globin modifiers on the phenotype and clinical severity of β-thalassemia. We first discussed the impact of the excess of free α-globin on the phenotype of β-thalassemia. We then reviewed studies focusing on the expression of α-globin-stabilizing protein (AHSP), as a potential strategy of counteracting the effects of the excess of free α-globin on erythroid cells. Alternative processes controlling α-globin excess were also considered, including the activation of autophagy by β-thalassemia erythroid cells. Altogether, the studies reviewed herein are expected to have a potential impact on the management of patients with β-thalassemia and other hemoglobinopathies for which reduction in α-globin excess is clinically beneficial. [ABSTRACT FROM AUTHOR]

Induction of fetal hemoglobin (HbF) is highly beneficial for patients carrying β-thalassemia, and novel HbF inducers are highly needed. Here, we describe a new class of promising HbF inducers characterized by an isoxazole chemical skeleton and obtained through modification of two natural molecules, geldanamycin and radicicol. After preliminary biological assays based on benzidine staining and RT-qPCR conducted on human erythroleukemic K562 cells, we employed erythroid precursors cells (ErPCs) isolated from β-thalassemic patients. ErPCs weretreated with appropriate concentrations of isoxazole derivatives. The accumulation of globin mRNAs was studied by RT-qPCR, and hemoglobin production by HPLC. We demonstrated the high efficacy of isozaxoles in inducing HbF. Most of these derivatives displayed an activity similar to that observed using known HbF inducers, such as hydroxyurea (HU) or rapamycin; some of the analyzed compounds were able to induce HbF with more efficiency than HU. All the compounds were active in reducing the excess of free α-globin in treated ErPCs. All the compounds displayed a lack of genotoxicity. These novel isoxazoles deserve further pre-clinical study aimed at verifying whether they are suitable for the development of therapeutic protocols for β-thalassemia. [ABSTRACT FROM AUTHOR]

The β-thalassemias are hereditary monogenic diseases characterized by a low or absent production of adult hemoglobin and excess in the content of α-globin. This excess is cytotoxic for the erythroid cells and responsible for the β-thalassemia-associated ineffective erythropoiesis. Therefore, the decrease in excess α-globin is a relevant clinical effect for these patients and can be realized through the induction of fetal hemoglobin, autophagy, or both. The in vivo effects of sirolimus (rapamycin) and analogs on the induction of fetal hemoglobin (HbF) are of key importance for therapeutic protocols in a variety of hemoglobinopathies, including β-thalassemias. In this research communication, we report data showing that a decrease in autophagy-associated p62 protein, increased expression of ULK-1, and reduction in excess α-globin are occurring in erythroid precursors (ErPCs) stimulated in vitro with low dosages of sirolimus. In addition, increased ULK-1 mRNA content and a decrease in α-globin content were found in ErPCs isolated from β-thalassemia patients recruited for the NCT03877809 clinical trial and treated with 0.5–2 mg/day sirolimus. Our data support the concept that autophagy, ULK1 expression, and α-globin chain reduction should be considered important endpoints in sirolimus-based clinical trials for β-thalassemias. [ABSTRACT FROM AUTHOR]

The anticancer drug mithramycin (MTH), has been proposed for drug repurposing after the finding that it is a potent inducer of fetal hemoglobin (HbF) production in erythroid precursor cells (ErPCs) from β-thalassemia patients. In this respect, previously published studies indicate that MTH is very active in inducing increased expression of γ-globin genes in erythroid cells. This is clinically relevant, as it is firmly established that HbF induction is a valuable approach for the therapy of β-thalassemia and for ameliorating the clinical parameters of sickle-cell disease (SCD). Therefore, the identification of MTH biochemical/molecular targets is of great interest. This study is inspired by recent robust evidence indicating that the expression of γ-globin genes is controlled in adult erythroid cells by different transcriptional repressors, including Oct4, MYB, BCL11A, Sp1, KLF3 and others. Among these, BCL11A is very important. In the present paper we report evidence indicating that alterations of BCL11A gene expression and biological functions occur during MTH-mediated erythroid differentiation. Our study demonstrates that one of the mechanisms of action of MTH is a down-regulation of the transcription of the BCL11A gene, while a second mechanism of action is the inhibition of the molecular interactions between the BCL11A complex and specific sequences of the γ-globin gene promoter. [ABSTRACT FROM AUTHOR]

Simple Summary: In this review article, we present the fascinating story of sirolimus (rapamycin), a drug known to be able to induce fetal hemoglobin, and for this reason of great interest for the treatment of β-thalassemia. In fact, high levels of fetal hemoglobin have been demonstrated to be beneficial for β-thalassemia patients. The story began in 1964, with METEI (Medical Expedition to Easter Island, Rapa Nui). During this expedition, samples of the soil from different parts of the island were collected and, from this material, an antibiotic-producing microorganism (Streptomyces hygroscopicus) was identified and rapamycin was extracted from the mycelium with organic solvents. The story continued with the finding that rapamycin was a very active anti-bacterial and anti-fungal agent. In addition, rapamycin was demonstrated to inhibit the cell growth of tumor cell lines. More importantly, rapamycin was found to be an immunosuppressive agent applicable to prevent kidney rejection after transplant. More recently, rapamycin was found to be a potent inducer of fetal hemoglobin both in vitro using cell lines, in vivo using experimental mice, and in patients treated with this compound. These studies were the basis for proposing clinical trials on β-thalassemia patients. In this review article, we present the fascinating story of rapamycin (sirolimus), a drug able to induce γ-globin gene expression and increased production of fetal hemoglobin (HbF) in erythroid cells, including primary erythroid precursor cells (ErPCs) isolated from β-thalassemia patients. For this reason, rapamycin is considered of great interest for the treatment of β-thalassemia. In fact, high levels of HbF are known to be highly beneficial for β-thalassemia patients. The story of rapamycin discovery began in 1964, with METEI, the Medical Expedition to Easter Island (Rapa Nui). During this expedition, samples of the soil from different parts of the island were collected and, from this material, an antibiotic-producing microorganism (Streptomyces hygroscopicus) was identified. Rapamycin was extracted from the mycelium with organic solvents, isolated, and demonstrated to be very active as an anti-bacterial and anti-fungal agent. Later, rapamycin was demonstrated to inhibit the in vitro cell growth of tumor cell lines. More importantly, rapamycin was found to be an immunosuppressive agent applicable to prevent kidney rejection after transplantation. More recently, rapamycin was found to be a potent inducer of HbF both in vitro using ErPCs isolated from β-thalassemia patients, in vivo using experimental mice, and in patients treated with this compound. These studies were the basis for proposing clinical trials on β-thalassemia patients. [ABSTRACT FROM AUTHOR]

The β-thalassemias are a group of monogenic hereditary hematological disorders caused by deletions and/or mutations of the β-globin gene, leading to low or absent production of adult hemoglobin (HbA). For β-thalassemia, sirolimus has been under clinical consideration in two trials (NCT03877809 and NCT04247750). A reduced immune response to anti-SARS-CoV-2 vaccination has been reported in organ recipient patients treated with the immunosuppressant sirolimus. Therefore, there was some concern regarding the fact that monotherapy with sirolimus would reduce the antibody response after SARS-CoV-2 vaccination. In the representative clinical case reported in this study, sirolimus treatment induced the expected increase of fetal hemoglobin (HbF) but did not prevent the production of anti-SARS-CoV-2 IgG after vaccination with mRNA-1273 (Moderna). In our opinion, this case report should stimulate further studies on β-thalassemia patients under sirolimus monotherapy in order to confirm the safety (or even the positive effects) of sirolimus with respect to the humoral response to anti-SARS-CoV-2 vaccination. In addition, considering the extensive use of sirolimus for the treatment of other human pathologies (for instance, in organ transplantation, systemic lupus erythematosus, autoimmune cytopenia, and lymphangioleiomyomatosis), this case report study might be of general interest, as large numbers of patients are currently under sirolimus treatment. [ABSTRACT FROM AUTHOR]

One of the most relevant pathophysiological hallmarks of β-thalassemia is the accumulation of toxic α-globin chains inside erythroid cells, which is responsible for their premature death (hemolysis). In this context, the availability of an experimental model system mimicking the excess in α-globin chain production is still lacking. The objective of the present study was to produce and characterize K562 cellular clones forced to produce high amounts of α-globin, in order to develop an experimental model system suitable for studies aimed at the reduction of the accumulation of toxic α-globin aggregates. In the present study, we produced and characterized K562 cellular clones that, unlike the original K562 cell line, stably produced high levels of α-globin protein. As expected, the obtained clones had a tendency to undergo apoptosis that was proportional to the accumulation of α-globin, confirming the pivotal role of α-globin accumulation in damaging erythroid cells. Interestingly, the obtained clones seemed to trigger autophagy spontaneously, probably to overcome the accumulation/toxicity of the α-globin. We propose this new model system for the screening of pharmacological agents able to activate the full program of autophagy to reduce α-globin accumulation, but the model may be also suitable for new therapeutical approaches targeted at the reduction of the expression of the α-globin gene. [ABSTRACT FROM AUTHOR]

Inhibitors of the mammalian target of rapamycin (mTOR) have been proposed to improve vaccine responses, especially in the elderly. Accordingly, testing mTOR inhibitors (such as Sirolimus) and other geroprotective drugs might be considered a key strategy to improve overall health resilience of aged populations. In this respect, Sirolimus (also known as rapamycin) is of great interest, in consideration of the fact that it is extensively used in routine therapy and in clinical studies for the treatment of several diseases. Recently, Sirolimus has been considered in laboratory and clinical studies aimed to find novel protocols for the therapy of hemoglobinopathies (e.g. β‐Thalassemia). The objective of the present study was to analyse the activity of CD4+ and CD8+ T cells in β‐Thalassemia patients treated with Sirolimus, taking advantages from the availability of cellular samples of the NCT03877809 clinical trial. The approach was to verify IFN‐γ releases following stimulation of peripheral blood mononuclear cells (PBMCs) to stimulatory CEF and CEFTA peptide pools, stimulatory for CD4+ and CD8+ T cells, respectively. The main results of the present study are that treatment of β‐Thalassemia patients with Sirolimus has a positive impact on the biological activity and number of memory CD4+ and CD8+ T cells releasing IFN‐γ following stimulation with antigenic stimuli present in immunological memory. These data are to our knowledge novel and in our opinion of interest, in consideration of the fact that β‐Thalassemia patients are considered prone to immune deficiency. [ABSTRACT FROM AUTHOR]

The human homologue of mouse Ly-1 antibody reactive clone protein (LYAR) is a putative novel regulator of γ-globin gene transcription. The LYAR DNA-binding motif (5′-GGTTAT-3′) is located within the 5′-UTR of the Aγ-globin gene. The LYAR rs368698783 (G>A) polymorphism is present in β-thalassemia patients and decreases the LYAR binding efficiency to the Aγ-globin gene. The objective of this study was to stratify β-thalassemia patients with respect to the rs368698783 (G>A) polymorphism and to verify whether their erythroid precursor cells (ErPCs) differentially respond in vitro to selected fetal hemoglobin (HbF) inducers. The rs368698783 (G>A) polymorphism was detected by DNA sequencing, hemoglobin production by HPLC, and accumulation of globin mRNAs by RT-qPCR. We found that the LYAR rs368698783 (G>A) polymorphism is associated with high basal and induced production of fetal hemoglobin in β-thalassemia patients. The most striking association was found using rapamycin as an HbF inducer. The results presented here could be considered important not only for basic biomedicine but also in applied translational research for precision medicine in personalized therapy of β-thalassemia. Accordingly, our data suggest that the rs368698783 polymorphism might be considered among the parameters useful to recruit patients with the highest probability of responding to in vivo hydroxyurea (HU) treatment. [ABSTRACT FROM AUTHOR]

Gene editing (GE) is an efficient strategy for correcting genetic mutations in monogenic hereditary diseases, including β-thalassemia. We have elsewhere reported that CRISPR-Cas9-based gene editing can be employed for the efficient correction of the β039-thalassemia mutation. On the other hand, robust evidence demonstrates that the increased production of fetal hemoglobin (HbF) can be beneficial for patients with β-thalassemia. The aim of our study was to verify whether the de novo production of adult hemoglobin (HbA) using CRISPR-Cas9 gene editing can be combined with HbF induction protocols. The gene editing of the β039-globin mutation was obtained using a CRISPR-Cas9-based experimental strategy; the correction of the gene sequence and the transcription of the corrected gene were analyzed by allele-specific droplet digital PCR and RT-qPCR, respectively; the relative content of HbA and HbF was studied by high-performance liquid chromatography (HPLC) and Western blotting. For HbF induction, the repurposed drug rapamycin was used. The data obtained conclusively demonstrate that the maximal production of HbA and HbF is obtained in GE-corrected, rapamycin-induced erythroid progenitors isolated from β039-thalassemia patients. In conclusion, GE and HbF induction might be used in combination in order to achieve the de novo production of HbA together with an increase in induced HbF. [ABSTRACT FROM AUTHOR]

Recent studies have identified and characterized a novel putative transcriptional repressor site in a 5′ untranslated region of the Aγ-globin gene that interacts with the Ly-1 antibody reactive clone (LYAR) protein. LYAR binds the 5'-GGTTAT-3' site of the Aγ-globin gene, and this molecular interaction causes repression of gene transcription. In β-thalassemia patients, a polymorphism has been demonstrated (the rs368698783 G>A polymorphism) within the 5′-GGTTAT-3′ LYAR-binding site of the Aγ-globin gene. The major results gathered from surface plasmon resonance based biospecific interaction analysis (SPR-BIA) studies (using crude nuclear extracts, LYAR-enriched lysates, and recombinant LYAR) support the concept that the rs368698783 G>A polymorphism of the Aγ-globin gene attenuates the efficiency of LYAR binding to the LYAR-binding site. This conclusion was fully confirmed by a molecular docking analysis. This might lead to a very important difference in erythroid cells from β-thalassemia patients in respect to basal and induced levels of production of fetal hemoglobin. The novelty of the reported SPR-BIA method is that it allows the characterization and validation of the altered binding of a key nuclear factor (LYAR) to mutated LYAR-binding sites. These results, in addition to theoretical implications, should be considered of interest in applied pharmacology studies as a basis for the screening of drugs able to inhibit LYAR–DNA interactions. This might lead to the identification of molecules facilitating induced increase of γ-globin gene expression and fetal hemoglobin production in erythroid cells, which is associated with possible reduction of the clinical severity of the β-thalassemia phenotype. [ABSTRACT FROM AUTHOR]

The involvement of microRNAs in the control of repressors of human γ-globin gene transcription has been firmly demonstrated, as described for the miR-486-3p mediated down-regulation of BCL11A. On the other hand, we have reported that miR-210 is involved in erythroid differentiation and, possibly, in γ-globin gene up-regulation. In the present study, we have identified the coding sequence of BCL11A as a possible target of miR-210. The following results sustain this hypothesis: (a) interactions between miR-210 and the miR-210 BCL11A site were demonstrated by SPR-based biomolecular interaction analysis (BIA); (b) the miR-210 site of BCL11A is conserved through molecular evolution; (c) forced expression of miR-210 leads to decrease of BCL11A-XL and increase of γ-globin mRNA content in erythroid cells, including erythroid precursors isolated from β-thalassemia patients. Our study suggests that the coding mRNA sequence of BCL11A can be targeted by miR-210. In addition to the theoretical point of view, these data are of interest from the applied point of view, supporting a novel strategy to inhibit BCL11A by mimicking miR-210 functions, accordingly with the concept supported by several papers and patent applications that inhibition of BCL11A is an efficient strategy for fetal hemoglobin induction in the treatment of β-thalassemia. [ABSTRACT FROM AUTHOR]

Background: Increase of the expression of γ-globin gene and high production of fetal hemoglobin (HbF) in β-thalassemia patients is widely accepted as associated with a milder or even asymptomatic disease. The search for HbF-associated polymorphisms (such as the XmnI, BCL11A and MYB polymorphisms) has recently gained great attention, in order to stratify β-thalassemia patients with respect to expectancy of the first transfusion, need for annual intake of blood, response to HbF inducers (the most studied of which is hydroxyurea). Methods: Aγ-globin gene sequencing was performed on genomic DNA isolated from a total of 75 β-thalassemia patients, including 31 β039/β039, 33 β039/β+IVSI-110, 9 β+IVSI-110/β+IVSI-110, one β0IVSI-1/β+IVSI-6 and one β039/β+IVSI-6. Results: The results show that the rs368698783 polymorphism is present in β-thalassemia patients in the 5'UTR sequence (+25) of the Aγ-globin gene, known to affect the LYAR (human homologue of mouse Ly-1 antibody reactive clone) binding site 5'-GGTTAT-3'. This Aγ(+25 G->A) polymorphism is associated with the Gγ-globin-XmnI polymorphism and both are linked with the β039-globin gene, but not with the β+IVSI-110-globin gene. In agreement with the expectation that this mutation alters the LYAR binding activity, we found that the Aγ(+25 G->A) and Gγ-globin-XmnI polymorphisms are associated with high HbF in erythroid precursor cells isolated from β039/β039 thalassemia patients. Conclusions: As a potential explanation of our findings, we hypothesize that in β-thalassemia the Gγ-globin-XmnI/Aγ-globin-(G->A) genotype is frequently under genetic linkage with β0-thalassemia mutations, but not with the β+-thalassemia mutation here studied (i.e. β+IVSI-110) and that this genetic combination has been selected within the population of β0-thalassemia patients, due to functional association with high HbF. Here we describe the characterization of the rs368698783 (+25 G->A) polymorphism of the Aγ-globin gene associated in β039 thalassemia patients with high HbF in erythroid precursor cells. [ABSTRACT FROM AUTHOR]

β-thalassemias are among the most common inherited hemoglobinopathies worldwide and are the result of autosomal mutations in the gene encoding β-globin, causing an absence or low-level production of adult hemoglobin (HbA). Induction of fetal hemoglobin (HbF) is considered to be of key importance for the development of therapeutic protocols for β-thalassemia and novel HbF inducers need to be proposed for pre-clinical development. The main purpose on this study was to analyze Cinchona alkaloids (cinchonidine, quinidine and cinchonine) as natural HbF-inducing agents in human erythroid cells. The analytical methods employed were Reverse Transcription quantitative real-time PCR (RT-qPCR) (for quantification of γ-globin mRNA) and High Performance Liquid Chromatography (HPLC) (for analysis of the hemoglobin pattern). After an initial analysis using the K562 cell line as an experimental model system, showing induction of hemoglobin and γ-globin mRNA, we verified whether the two more active compounds, cinchonidine and quinidine, were able to induce HbF in erythroid progenitor cells isolated from β-thalassemia patients. The data obtained demonstrate that cinchonidine and quinidine are potent inducers of γ-globin mRNA and HbF in erythroid progenitor cells isolated from nine β-thalassemia patients. In addition, both compounds were found to synergize with the HbF inducer sirolimus for maximal production of HbF. The data obtained strongly indicate that these compounds deserve consideration in the development of pre-clinical approaches for therapeutic protocols of β-thalassemia. [ABSTRACT FROM AUTHOR]

The aim of the present study was to identify molecular analogs of angelicin (ANG) able to increase erythroid differentiation of K562 cells and expression of gamma-globin genes in human erythroid precursor cells, with low effects on apoptosis. ANG-like molecules are well-known photosensitizers largely used for their antiproliferative activity in the treatment of different skin diseases (i.e., psoriasis, vitiligo, eczema, and mycosis fungoides). To verify the activity of these derivatives, we employed three experimental cell systems: (1) the human leukemic K562 cell line, (2) K562 cell clones stably transfected with a pCCL construct carrying green-EGFP under the gamma-globin gene promoter, and (3) the two-phase liquid culture of human erythroid progenitors isolated from normal donors and beta-thalassemia patients. The results of our study suggest that trimethyl ANG is a powerful inducer of erythroid differentiation, compared with known inducers, such as ANG, cytosine arabinoside, mithramycin, and cisplatin. These data could have practical relevance, because pharmacologically mediated regulation of human gamma-globin gene expression, with the consequent induction of fetal hemoglobin, is considered a potential therapeutic approach in hematological disorders including beta-thalassemia and sickle cell anemia. [ABSTRACT FROM AUTHOR]

We report in this paper a novel thalassemia mutation (insertion of a single A nucleotide within the exon 1, at codon 18, of the β-globin gene) associated with a deletion of the δβ-globin gene region, in a patient exhibiting high persistence of fetal hemoglobin. The novel mutation causes a frameshift with the generation of a UGA stop codon. Analysis of the parent’s DNA demonstrates that the A insertion and frameshift mutation are inherited from the father, while the δβ-globin gene deletion is inherited from the mother. Gene dosage analysis and deletion-specific PCR demonstrate that the deletion is the (δβ)0 Sicilian deletion, involving a 13.4-kb δβ-globin gene region. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Optical biosensors based on Surface Plasmon Resonance (SPR), such as the Biacore™ X100, are widely used to study in real-time and in label-free mode bio-molecular interactions, including those allowing the identification of single point mutations responsible of genetic diseases, such as thalassemia and cystic fibrosis. The aim of this study was to verify whether the Biacore™ X100 can be proposed for the real-time detection of four mutations of the human β-globin gene causing β-thalassemia, a genetic blood disorder associated with absence (β 0 ) or reduction (β + ) of adult hemoglobin and severe anemia. In particular we analyzed the most frequent thalassemia point mutations present in the Mediterranean area (β 0 39, β 0 IVSI-1, β + IVSI-110 and β + IVSI-6) using a novel SPR-based interaction format where two oligonucleotide probes (one complementary to the normal sequence and the other to the mutated one) were immobilized on sensor chips and asymmetric PCR targets obtained from genomic DNA of analyzed subjects were injected. For the development of the diagnostic approach, genomic DNAs of different genotypes for each mutation were obtained from blood samples or salivary swabs of 71 subjects, including healthy individuals, heterozygous β-thalassemia carriers and homozygous β-thalassemia patients. The results obtained allow proposing a new non-invasive diagnostic SPR-based protocol for thalassemia single point mutations using blood samples and salivary swabs as a source of genomic DNA. [ABSTRACT FROM AUTHOR]