A Rational Approach to Drug Repositioning in β-thalassemia: Induction of Fetal Hemoglobin by Established Drugs

Drug repositioning and the relevance of orphan drug designation for β-thalassemia is reviewed. Drug repositioning and similar terms ('drug repurposing', 'drug reprofiling', 'drug redirecting', 'drug rescue', 'drug re-tasking' and/or 'drug rediscovery') have gained great attention, especially in the field or rare diseases (RDs), and represent relevant novel drug development strategies to be considered together with the 'off-label' use of pharmaceutical products under clinical trial regimen. The most significant advantage of drug repositioning over traditional drug development is that the repositioned drug has already passed a significant number of short- and long-term toxicity tests, as well as it has already undergone pharmacokinetic and pharmacodynamic (PK/PD) studies. The established safety of repositioned drugs is known to significantly reduce the probability of project failure. Furthermore, development of repurposed drugs can shorten much of the time needed to bring a drug to market. Finally, patent filing of repurposed drugs is expected to catch the attention of pharmaceutical industries interested in the development of therapeutic protocols for RDs. Repurposed molecules that could be proposed as potential drugs for β-thalassemia, will be reported, with some of the most solid examples, including sirolimus (rapamycin) that recently has been tested in a pilot clinical trial

BCL11A mRNA targeting by miR-210: A possible network regulating γ-globin gene expression

The involvement of microRNAs in the control of repressors of human g-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

An Aγ-globin G->A gene polymorphism associated with β(0)39 thalassemia globin gene and high fetal hemoglobin production

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)

A validated cellular biobank for β-thalassemia

Background: Cellular biobanking is a key resource for collaborative networks planning to use same cells in studies aimed at solving a variety of biological and biomedical issues. This approach is of great importance in studies on β-thalassemia, since the recruitment of patients and collection of specimens can represent a crucial and often limiting factor in the experimental planning. Methods: Erythroid precursor cells were obtained from 72 patients, mostly β-thalassemic, expanded and cryopreserved. Expression of globin genes was analyzed by real time RT-qPCR. Hemoglobin production was studied by HPLC. Results: In this paper we describe the production and validation of a Thal-Biobank constituted by expanded erythroid precursor cells from β-thalassemia patients. The biobanked samples were validated for maintenance of their phenotype after (a) cell isolation from same patients during independent phlebotomies, (b) freezing step in different biobanked cryovials, (c) thawing step and analysis at different time points. Reproducibility was confirmed by shipping the frozen biobanked cells to different laboratories, where the cells were thawed, cultured and analyzed using the same standardized procedures. The biobanked cells were stratified on the basis of their baseline level of fetal hemoglobin production and exposed to fetal hemoglobin inducers. Conclusion: The use of biobanked cells allows stratification of the patients with respect to fetal hemoglobin production and can be used for determining the response to the fetal hemoglobin inducer hydroxyurea and to gene therapy protocols with reproducible results

Expression of γ-globin genes in β-thalassemia patients treated with sirolimus: results from a pilot clinical trial (Sirthalaclin)

Introduction: β-thalassemia is caused by autosomal mutations in the β-globin gene, which induce the absence or low-level synthesis of β-globin in erythroid cells. It is widely accepted that a high production of fetal hemoglobin (HbF) is beneficial for patients with β-thalassemia. Sirolimus, also known as rapamycin, is a lipophilic macrolide isolated from a strain of Streptomyces hygroscopicus that serves as a strong HbF inducer in vitro and in vivo. In this study, we report biochemical, molecular, and clinical results of a sirolimus-based NCT03877809 clinical trial (a personalized medicine approach for β-thalassemia transfusion-dependent patients: testing sirolimus in a first pilot clinical trial, Sirthalaclin). Methods: Accumulation of γ-globin mRNA was analyzed using reverse-transcription quantitative polymerase chain reaction (PCR), while the hemoglobin pattern was analyzed using high-performance liquid chromatography (HPLC). The immunophenotype was analyzed using a fluorescence-activated cell sorter (FACS), with antibodies against CD3, CD4, CD8, CD14, CD19, CD25 (for analysis of peripheral blood mononuclear cells), or CD71 and CD235a (for analysis of in vitro cultured erythroid precursors). Results: The results were obtained in eight patients with the β+/β+ and β+/β0 genotypes, who were treated with a starting dosage of 1 mg/day sirolimus for 24–48 weeks. The first finding of this study was that the expression of γ-globin mRNA increased in the blood and erythroid precursor cells isolated from β-thalassemia patients treated with low-dose sirolimus. This trial also led to the important finding that sirolimus influences erythropoiesis and reduces biochemical markers associated with ineffective erythropoiesis (excess free α-globin chains, bilirubin, soluble transferrin receptor, and ferritin). A decrease in the transfusion demand index was observed in most (7/8) of the patients. The drug was well tolerated, with minor effects on the immunophenotype, and an only side effect of frequently occurring stomatitis. Conclusion: The data obtained indicate that low doses of sirolimus modify hematopoiesis and induce increased expression of γ-globin genes in a subset of patients with β-thalassemia. Further clinical trials are warranted, possibly including testing of the drug in patients with less severe forms of the disease and exploring combination therapies. © The Author(s), 2022

Fetal Hemoglobin Inducers from the Natural World: A Novel Approach for Identification of Drugs for the Treatment of β-Thalassemia and Sickle-Cell Anemia

The objective of this review is to present examples of lead compounds identified from biological material (fungi, plant extracts and agro-industry material) and of possible interest in the field of a pharmacological approach to the therapy of β-thalassemia using molecules able to stimulate production of fetal hemoglobin (HbF) in adults. Concerning the employment of HbF inducers as potential drugs for pharmacological treatment of β-thalassemia, the following conclusions can be reached: (i) this therapeutic approach is reasonable, on the basis of the clinical parameters exhibited by hereditary persistence of fetal hemoglobin patients, (ii) clinical trials (even if still limited) employing HbF inducers were effective in ameliorating the symptoms of β-thalassemia patients, (iii) good correlation of in vivo and in vitro results of HbF synthesis and γ-globin mRNA accumulation indicates that in vitro testing might be predictive of in vivo responses and (iv) combined use of different inducers might be useful to maximize HbF, both in vitro and in vivo. In this review, we present three examples of HbF inducers from the natural world: (i) angelicin and linear psoralens, contained in plant extracts from Angelica arcangelica and Aegle marmelos, (ii) resveratrol, a polyphenol found in grapes and several plant extracts and (iii) rapamycin, isolated from Streptomyces hygroscopicus

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5–11 December, to 17.5% (25/143 samples) in the week 12–18, to 65.9% (89/135 samples) in the week 19–25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased fromone in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5-11 December, to 17.5% (25/143 samples) in the week 12-18, to 65.9% (89/135 samples) in the week 19-25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased from one in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons. In conclusion, we designed an RT-qPCR assay capable to detect the Omicron variant, which can be successfully used for the purpose of wastewater-based epidemiology. We also described the history of the introduction and diffusion of the Omicron variant in the Italian population and territory, confirming the effectiveness of sewage monitoring as a powerful surveillance tool

Recent trends in the gene therapy of β-thalassemia

The β-thalassemias are a group of hereditary hematological diseases caused by over 300 mutations of the adult β-globin gene. Together with sickle cell anemia, thalassemia syndromes are among the most impactful diseases in developing countries, in which the lack of genetic counseling and prenatal diagnosis have contributed to the maintenance of a very high frequency of these genetic diseases in the population. Gene therapy for β-thalassemia has recently seen steadily accelerating progress and has reached a crossroads in its development. Presently, data from past and ongoing clinical trials guide the design of further clinical and preclinical studies based on gene augmentation, while fundamental insights into globin switching and new technology developments have inspired the investigation of novel gene-therapy approaches. Moreover, human erythropoietic stem cells from β-thalassemia patients have been the cellular targets of choice to date whereas future gene-therapy studies might increasingly draw on induced pluripotent stem cells. Herein, we summarize the most significant developments in β-thalassemia gene therapy over the last decade, with a strong emphasis on the most recent findings, for β-thalassemia model systems; for β-, γ-, and anti-sickling β-globin gene addition and combinatorial approaches including the latest results of clinical trials; and for novel approaches, such as transgene-mediated activation of γ-globin and genome editing using designer nucleases