Increased Expression of α-Hemoglobin Stabilizing Protein (AHSP) mRNA in Erythroid Precursor Cells Isolated from ß-Thalassemia Patients Treated with Sirolimus (Rapamycin).

Background/Objectives: in ß-thalassemia, important clinical complications are caused by the presence of free α-globin chains in the erythroid cells of ß-thalassemia patients. These free α-globin chains are present in excess as a result of the lack of ß-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 ß-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 ß-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 ß-thalassemia patients in comparison to ErPCs isolated from healthy subjects. In addition, AHSP expression was further induced by treatment of ß-thalassemia ErPCs with sirolimus. Finally, AHSP mRNA was expressed at an increased level in ErPCs of sirolimus-treated ß-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.

Impact of α-Globin Gene Expression and α-Globin Modifiers on the Phenotype of ß-Thalassemia and Other Hemoglobinopathies: Implications for Patient Management.

In this short review, we presented and discussed studies on the expression of globin genes in ß-thalassemia, focusing on the impact of α-globin gene expression and α-globin modifiers on the phenotype and clinical severity of ß-thalassemia. We first discussed the impact of the excess of free α-globin on the phenotype of ß-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 ß-thalassemia erythroid cells. Altogether, the studies reviewed herein are expected to have a potential impact on the management of patients with ß-thalassemia and other hemoglobinopathies for which reduction in α-globin excess is clinically beneficial.

Inhibitory effects of SARS-CoV-2 spike protein and BNT162b2 vaccine on erythropoietin-induced globin gene expression in erythroid precursor cells from patients with ß-thalassemia.

During the recent coronavirus disease 2019 (COVID-19) pandemic several patients with ß-thalassemia have been infected by severe acute respiratory syndrome coronavirus (SARS-CoV-2), and most patients were vaccinated against SARS-CoV-2. Recent studies demonstrate an impact of SARS-CoV-2 infection on the hematopoietic system. The main objective of this study was to verify the effects of exposure of erythroid precursor cells (ErPCs) from patients with ß-thalassemia to SARS-CoV-2 spike protein (S-protein) and the BNT162b2 vaccine. Erythropoietin (EPO)-cultured ErPCs have been either untreated or treated with S-protein or BNT162b2 vaccine. The employed ErPCs were from a ß-thalassemia cellular Biobank developed before the COVID-19 pandemic. The genotypes were ß+-IVSI-110/ß+-IVSI-110 (one patient),  ß039/ß+-IVSI-110 (3 patients), and ß039/ ß039 (2 patients). After treatment with S-protein or BNT162b2 for 5 days, lysates were analyzed by high performance liquid chromatography (HPLC), for hemoglobin production, and isolated RNA was assayed by RT-qPCR, for detection of globin gene expression. The main conclusions of the results obtained are that SARS-CoV-2 S-protein and BNT162b2 vaccine (a) inhibit fetal hemoglobin (HbF) production by ß-thalassemic ErPCs and (b) inhibit γ-globin mRNA accumulation. In addition, we have performed in silico studies suggesting a high affinity of S-protein to HbF. Remarkably, the binding interaction energy of fetal hemoglobin to S-protein was comparable with that of angiotensin-converting enzyme 2 (ACE2). Our results are consistent with the hypothesis of a relevant impact of SARS-CoV-2 infection and COVID-19 vaccination on the hematopoietic system.

The anti-SARS-CoV-2 BNT162b2 vaccine suppresses mithramycin-induced erythroid differentiation and expression of embryo-fetal globin genes in human erythroleukemia K562 cells.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causative of the ongoing coronavirus disease 2019 (COVID-19) pandemic. The SARS-CoV-2 Spike protein (S-protein) plays an important role in the early phase of SARS-CoV-2 infection through efficient interaction with ACE2. The S-protein is produced by RNA-based COVID-19 vaccines, that were fundamental for the reduction of the viral spread within the population and the clinical severity of COVID-19. However, the S-protein has been hypothesized to be responsible for damaging cells of several tissues and for some important side effects of RNA-based COVID-19 vaccines. Considering the impact of COVID-19 and SARS-CoV-2 infection on the hematopoietic system, the aim of this study was to verify the effect of the BNT162b2 vaccine on erythroid differentiation of the human K562 cell line, that has been in the past intensively studied as a model system mimicking some steps of erythropoiesis. In this context, we focused on hemoglobin production and induced expression of embryo-fetal globin genes, that are among the most important features of K562 erythroid differentiation. We found that the BNT162b2 vaccine suppresses mithramycin-induced erythroid differentiation of K562 cells. Reverse-transcription-qPCR and Western blotting assays demonstrated that suppression of erythroid differentiation was associated with sharp inhibition of the expression of α-globin and γ-globin mRNA accumulation. Inhibition of accumulation of ζ-globin and ε-globin mRNAs was also observed. In addition, we provide in silico studies suggesting a direct interaction between SARS-CoV-2 Spike protein and Hb Portland, that is the major hemoglobin produced by K562 cells. This study thus provides information suggesting the need of great attention on possible alteration of hematopoietic parameters following SARS-CoV-2 infection and/or COVID-19 vaccination.

Decrease in α-Globin and Increase in the Autophagy-Activating Kinase ULK1 mRNA in Erythroid Precursors from ß-Thalassemia Patients Treated with Sirolimus.

The ß-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 ß-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 ß-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 ß-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 ß-thalassemias.

Effects of Mithramycin on BCL11A Gene Expression and on the Interaction of the BCL11A Transcriptional Complex to γ-Globin Gene Promoter Sequences.

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 ß-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 ß-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.

The Long Scientific Journey of Sirolimus (Rapamycin): From the Soil of Easter Island (Rapa Nui) to Applied Research and Clinical Trials on ß-Thalassemia and Other Hemoglobinopathies.

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 ß-thalassemia patients. For this reason, rapamycin is considered of great interest for the treatment of ß-thalassemia. In fact, high levels of HbF are known to be highly beneficial for ß-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 ß-thalassemia patients, in vivo using experimental mice, and in patients treated with this compound. These studies were the basis for proposing clinical trials on ß-thalassemia patients.

Combined approaches for increasing fetal hemoglobin (HbF) and de novo production of adult hemoglobin (HbA) in erythroid cells from ß-thalassemia patients: treatment with HbF inducers and CRISPR-Cas9 based genome editing.

Genome editing (GE) is one of the most efficient and useful molecular approaches to correct the effects of gene mutations in hereditary monogenetic diseases, including ß-thalassemia. CRISPR-Cas9 gene editing has been proposed for effective correction of the ß-thalassemia mutation, obtaining high-level "de novo" production of adult hemoglobin (HbA). In addition to the correction of the primary gene mutations causing ß-thalassemia, several reports demonstrate that gene editing can be employed to increase fetal hemoglobin (HbF), obtaining important clinical benefits in treated ß-thalassemia patients. This important objective can be achieved through CRISPR-Cas9 disruption of genes encoding transcriptional repressors of γ-globin gene expression (such as BCL11A, SOX6, KLF-1) or their binding sites in the HBG promoter, mimicking non-deletional and deletional HPFH mutations. These two approaches (ß-globin gene correction and genome editing of the genes encoding repressors of γ-globin gene transcription) can be, at least in theory, combined. However, since multiplex CRISPR-Cas9 gene editing is associated with documented evidence concerning possible genotoxicity, this review is focused on the possibility to combine pharmacologically-mediated HbF induction protocols with the "de novo" production of HbA using CRISPR-Cas9 gene editing.

Cationic Calix[4]arene Vectors to Efficiently Deliver AntimiRNA Peptide Nucleic Acids (PNAs) and miRNA Mimics.

One of the most appealing approaches for regulating gene expression, named the "microRNA therapeutic" method, is based on the regulation of the activity of microRNAs (miRNAs), the intracellular levels of which are dysregulated in many diseases, including cancer. This can be achieved by miRNA inhibition with antimiRNA molecules in the case of overexpressed microRNAs, or by using miRNA-mimics to restore downregulated microRNAs that are associated with the target disease. The development of new efficient, low-toxic, and targeted vectors of such molecules represents a key topic in the field of the pharmacological modulation of microRNAs. We compared the delivery efficiency of a small library of cationic calix[4]arene vectors complexed with fluorescent antimiRNA molecules (Peptide Nucleic Acids, PNAs), pre-miRNA (microRNA precursors), and mature microRNAs, in glioma- and colon-cancer cellular models. The transfection was assayed by cytofluorimetry, cell imaging assays, and RT-qPCR. The calix[4]arene-based vectors were shown to be powerful tools to facilitate the uptake of both neutral (PNAs) and negatively charged (pre-miRNAs and mature microRNAs) molecules showing low toxicity in transfected cells and ability to compete with commercially available vectors in terms of delivery efficiency. These results could be of great interest to validate microRNA therapeutics approaches for future application in personalized treatment and precision medicine.

Effects of Sulforaphane on SARS­CoV­2 infection and NF­κB dependent expression of genes involved in the COVID­19 'cytokine storm'.

Since its spread at the beginning of 2020, the coronavirus disease 2019 (COVID­19) pandemic represents one of the major health problems. Despite the approval, testing, and worldwide distribution of anti­severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) vaccines, the development of specific antiviral agents targeting the SARS­CoV­2 life cycle with high efficiency, and/or interfering with the associated 'cytokine storm', is highly required. A recent study, conducted by the authors' group indicated that sulforaphane (SFN) inhibits the expression of IL­6 and IL­8 genes induced by the treatment of IB3­1 bronchial cells with a recombinant spike protein of SARS­CoV­2. In the present study, the ability of SFN to inhibit SARS­CoV­2 replication and the expression of pro­inflammatory genes encoding proteins of the COVID­19 'cytokine storm' was evaluated. SARS­CoV­2 replication was assessed in bronchial epithelial Calu­3 cells. Moreover, SARS­CoV­2 replication and expression of pro­inflammatory genes was evaluated by reverse transcription quantitative droplet digital PCR. The effects on the expression levels of NF­κB were assessed by western blotting. Molecular dynamics simulations of NF­kB/SFN interactions were conducted with Gromacs 2021.1 software under the Martini 2 CG force field. Computational studies indicated that i) SFN was stably bound with the NF­κB monomer; ii) a ternary NF­kB/SFN/DNA complex was formed; iii) the SFN interacted with both the protein and the nucleic acid molecules modifying the binding mode of the latter, and impairing the full interaction between the NF­κB protein and the DNA molecule. This finally stabilized the inactive complex. Molecular studies demonstrated that SFN i) inhibits the SARS­CoV­2 replication in infected Calu­3 cells, decreasing the production of the N­protein coding RNA sequences, ii) decreased NF­κB content in SARS­CoV­2 infected cells and inhibited the expression of NF­kB­dependent IL­1ß and IL­8 gene expression. The data obtained in the present study demonstrated inhibitory effects of SFN on the SARS­CoV­2 life cycle and on the expression levels of the pro­inflammatory genes, sustaining the possible use of SFN in the management of patients with COVID­19.

Effects of Sirolimus Treatment on Fetal Hemoglobin Production and Response to SARS-CoV-2 Vaccination: A Case Report Study.

The ß-thalassemias are a group of monogenic hereditary hematological disorders caused by deletions and/or mutations of the ß-globin gene, leading to low or absent production of adult hemoglobin (HbA). For ß-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 ß-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.

Combined Treatment of Cancer Cells Using Allyl Palladium Complexes Bearing Purine-Based NHC Ligands and Molecules Targeting MicroRNAs miR-221-3p and miR-222-3p: Synergistic Effects on Apoptosis.

Combined treatments employing lower concentrations of different drugs are used and studied to develop new and more effective anticancer therapeutic approaches. The combination therapy could be of great interest in the controlling of cancer. Regarding this, our research group has recently shown that peptide nucleic acids (PNAs) that target miR-221 are very effective and functional in inducing apoptosis of many tumor cells, including glioblastoma and colon cancer cells. Moreover, in a recent paper, we described a series of new palladium allyl complexes showing a strong antiproliferative activity on different tumor cell lines. The present study was aimed to analyze and validate the biological effects of the most active compounds tested, in combination with antagomiRNA molecules targeting two miRNAs, miR-221-3p and miR-222-3p. The obtained results show that a "combination therapy", produced by combining the antagomiRNAs targeting miR-221-3p, miR-222-3p and the palladium allyl complex 4d, is very effective in inducing apoptosis, supporting the concept that the combination treatment of cancer cells with antagomiRNAs targeting a specific upregulated oncomiRNAs (in this study miR-221-3p and miR-222-3p) and metal-based compounds represents a promising therapeutic strategy to increase the efficacy of the antitumor protocol, reducing side effects at the same time.

The Cystic Fibrosis Transmembrane Conductance Regulator Gene (CFTR) Is under Post-Transcriptional Control of microRNAs: Analysis of the Effects of agomiRNAs Mimicking miR-145-5p, miR-101-3p, and miR-335-5p.

(1) Background: MicroRNAs are involved in the expression of the gene encoding the chloride channel CFTR (Cystic Fibrosis Transmembrane Conductance Regulator); the objective of this short report is to study the effects of the treatment of bronchial epithelial Calu-3 cells with molecules mimicking the activity of pre-miR-145-5p, pre-miR-335-5p, and pre-miR-101-3p, and to discuss possible translational applications of these molecules in pre-clinical studies focusing on the development of protocols of possible interest in therapy; (2) Methods: CFTR mRNA was quantified by Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR). The production of the CFTR protein was assessed by Western blotting; (3) Results: The treatment of Calu-3 cells with agomiR-145-5p caused the highest inhibition of CFTR mRNA accumulation and CFTR production; (4) Conclusions: The treatment of target cells with the agomiR pre-miR-145-5p should be considered when CFTR gene expression should be inhibited in pathological conditions, such as polycystic kidney disease (PKD), some types of cancer, cholera, and SARS-CoV-2 infection.

Production and Characterization of K562 Cellular Clones Hyper-Expressing the Gene Encoding α-Globin: Preliminary Analysis of Biomarkers Associated with Autophagy.

One of the most relevant pathophysiological hallmarks of ß-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.

The rs368698783 (G>A) Polymorphism Affecting LYAR Binding to the Aγ-Globin Gene Is Associated with High Fetal Hemoglobin (HbF) in ß-Thalassemia Erythroid Precursor Cells Treated with HbF Inducers

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 ß-thalassemia patients and decreases the LYAR binding efficiency to the Aγ-globin gene. The objective of this study was to stratify ß-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 ß-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 ß-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.

SARS-CoV-2 viral entry and replication is impaired in Cystic Fibrosis airways due to ACE2 downregulation.

As an inherited disorder characterized by severe pulmonary disease, cystic fibrosis could be considered a comorbidity for coronavirus disease 2019. Instead, current clinical evidence seems to be heading in the opposite direction. To clarify whether host factors expressed by the Cystic Fibrosis epithelia may influence coronavirus disease 2019 progression, here we describe the expression of SARS-CoV-2 receptors in primary airway epithelial cells. We show that angiotensin converting enzyme 2 (ACE2) expression and localization are regulated by Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Consistently, our results indicate that dysfunctional CFTR channels alter susceptibility to SARS-CoV-2 infection, resulting in reduced viral entry and replication in Cystic Fibrosis cells. Depending on the pattern of ACE2 expression, the SARS-CoV-2 spike (S) protein induced high levels of Interleukin 6 in healthy donor-derived primary airway epithelial cells, but a very weak response in primary Cystic Fibrosis cells. Collectively, these data support that Cystic Fibrosis condition may be at least partially protecting from SARS-CoV-2 infection.

Effects of Sirolimus treatment on patients with ß-Thalassemia: Lymphocyte immunophenotype and biological activity of memory CD4+ and CD8+ T cells.

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. ß-Thalassemia). The objective of the present study was to analyse the activity of CD4+ and CD8+ T cells in ß-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 ß-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 ß-Thalassemia patients are considered prone to immune deficiency.

New Synthetic Isoxazole Derivatives Acting as Potent Inducers of Fetal Hemoglobin in Erythroid Precursor Cells Isolated from ß-Thalassemic Patients.

Induction of fetal hemoglobin (HbF) is highly beneficial for patients carrying ß-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 ß-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 ß-thalassemia.

Co-Treatment of Erythroid Cells from ß-Thalassemia Patients with CRISPR-Cas9-Based ß039-Globin Gene Editing and Induction of Fetal Hemoglobin.

Gene editing (GE) is an efficient strategy for correcting genetic mutations in monogenic hereditary diseases, including ß-thalassemia. We have elsewhere reported that CRISPR-Cas9-based gene editing can be employed for the efficient correction of the ß039-thalassemia mutation. On the other hand, robust evidence demonstrates that the increased production of fetal hemoglobin (HbF) can be beneficial for patients with ß-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 ß039-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 ß039-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.

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

Drug repositioning and the relevance of orphan drug designation for ß-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 ß-thalassemia, will be reported, with some of the most solid examples, including sirolimus (rapamycin) that recently has been tested in a pilot clinical trial.