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.

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.

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.

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.

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.

Synergistic Effects of A Combined Treatment of Glioblastoma U251 Cells with An Anti-miR-10b-5p Molecule and An AntiCancer Agent Based on 1-(3',4',5'-Trimethoxyphenyl)-2-Aryl-1H-Imidazole Scaffold.

(1) Background: In the development of new and more effective anticancer approaches, combined treatments appear of great interest. Combination therapy could be of importance in the management of glioblastoma (GBM), a lethal malignancy that accounts for 42% of cancer of the central nervous system, with a median survival of 15 months. This study aimed to verify the activity on a glioblastoma cancer cell line of one of the most active compounds of a novel series of tubulin polymerization inhibitors based on the 1-(3',4',5'-trimethoxyphenyl)-2-aryl-1H-imidazole scaffold, used in combination with a miRNA inhibitor molecule targeting the oncomiRNA miR-10b-5p. This microRNA was selected in consideration of the role of miR-10b-5p on the onset and progression of glioblastoma. (2) Methods: Apoptosis was analyzed by Annexin-V and Caspase 3/7 assays, efficacy of the anti-miR-10b-5p was assessed by determining the miR-10b-5p content by RT-qPCR. (3) Results: The results obtained show that a "combination therapy" performed by combining the use of an anti-miR-10b-5p and a 1-(3',4',5'-trimethoxyphenyl)-2-aryl-1H-imidazole derivative is an encouraging strategy to boost the efficacy of anticancer therapies and at the same time to reduce side effects.

Combined Treatment of Bronchial Epithelial Calu-3 Cells with Peptide Nucleic Acids Targeting miR-145-5p and miR-101-3p: Synergistic Enhancement of the Expression of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene encodes for a chloride channel defective in Cystic Fibrosis (CF). Accordingly, upregulation of its expression might be relevant for the development of therapeutic protocols for CF. MicroRNAs are deeply involved in the CFTR regulation and their targeting with miRNA inhibitors (including those based on Peptide Nucleic Acids, PNAs)is associated with CFTR upregulation. Targeting of miR-145-5p, miR-101-3p, and miR-335-5p with antisense PNAs was found to be associated with CFTR upregulation. The main objective of this study was to verify whether combined treatments with the most active PNAs are associated with increased CFTR gene expression. The data obtained demonstrate that synergism of upregulation of CFTR production can be obtained by combined treatments of Calu-3 cells with antisense PNAs targeting CFTR-regulating microRNAs. In particular, highly effective combinations were found with PNAs targeting miR-145-5p and miR-101-3p. Content of mRNAs was analyzed by RT-qPCR, the CFTR production by Western blotting. Combined treatment with antagomiRNAs might lead to maximized upregulation of CFTR and should be considered in the development of protocols for CFTR activation in pathological conditions in which CFTR gene expression is lacking, such as Cystic Fibrosis.

Treatment of Human Glioblastoma U251 Cells with Sulforaphane and a Peptide Nucleic Acid (PNA) Targeting miR-15b-5p: Synergistic Effects on Induction of Apoptosis.

Glioblastoma multiforme (GBM) is a lethal malignant tumor accounting for 42% of the tumors of the central nervous system, the median survival being 15 months. At present, no curative treatment is available for GBM and new drugs and therapeutic protocols are urgently needed. In this context, combined therapy appears to be a very interesting approach. The isothiocyanate sulforaphane (SFN) has been previously shown to induce apoptosis and inhibit the growth and invasion of GBM cells. On the other hand, the microRNA miR-15b is involved in invasiveness and proliferation in GBM and its inhibition is associated with the induction of apoptosis. On the basis of these observations, the objective of the present study was to determine whether a combined treatment using SFN and a peptide nucleic acid interfering with miR-15b-5p (PNA-a15b) might be proposed for increasing the pro-apoptotic effects of the single agents. To verify this hypothesis, we have treated GMB U251 cells with SFN alone, PNA-a15b alone or their combination. The cell viability, apoptosis and combination index were, respectively, analyzed by calcein staining, annexin-V and caspase-3/7 assays, and RT-qPCR for genes involved in apoptosis. The efficacy of the PNA-a15b determined the miR-15b-5p content analyzed by RT-qPCR. The results obtained indicate that SFN and PNA-a15b synergistically act in inducing the apoptosis of U251 cells. Therefore, the PNA-a15b might be proposed in a "combo-therapy" associated with SFN. Overall, this study suggests the feasibility of using combined treatments based on PNAs targeting miRNA involved in GBM and nutraceuticals able to stimulate apoptosis.

Treatment of Erythroid Precursor Cells from ß-Thalassemia Patients with Cinchona Alkaloids: Induction of Fetal Hemoglobin Production.

ß-thalassemias are among the most common inherited hemoglobinopathies worldwide and are the result of autosomal mutations in the gene encoding ß-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 ß-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 ß-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 ß-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 ß-thalassemia.

Synthesis and Biological Evaluation of New Antitubulin Agents Containing 2-(3',4',5'-trimethoxyanilino)-3,6-disubstituted-4,5,6,7-tetrahydrothieno[2,3-c]pyridine Scaffold.

Two novel series of compounds based on the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine and 4,5,6,7-tetrahydrobenzo[b]thiophene molecular skeleton, characterized by the presence of a 3',4',5'-trimethoxyanilino moiety and a cyano or an alkoxycarbonyl group at its 2- or 3-position, respectively, were designed, synthesized, and evaluated for antiproliferative activity on a panel of cancer cell lines and for selected highly active compounds, inhibition of tubulin polymerization, and cell cycle effects. We have identified the 2-(3',4',5'-trimethoxyanilino)-3-cyano-6-methoxycarbonyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine derivative 3a and its 6-ethoxycarbonyl homologue 3b as new antiproliferative agents that inhibit cancer cell growth with IC50 values ranging from 1.1 to 4.7 µM against a panel of three cancer cell lines. Their interaction with tubulin at micromolar levels leads to the accumulation of cells in the G2/M phase of the cell cycle and to an apoptotic cell death. The cell apoptosis study found that compounds 3a and 3b were very effective in the induction of apoptosis in a dose-dependent manner. These two derivatives did not induce cell death in normal human peripheral blood mononuclear cells, suggesting that they may be selective against cancer cells. Molecular docking studies confirmed that the inhibitory activity of these molecules on tubulin polymerization derived from binding to the colchicine site.

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.

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.

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.

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.

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.

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.

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 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.

Possible effects of sirolimus treatment on the long­term efficacy of COVID­19 vaccination in patients with ß­thalassemia: A theoretical perspective.

The pandemic caused by the severe acute respiratory syndrome coronavirus (SARS­CoV­2), responsible for coronavirus disease 2019 (COVID­19) has posed a major challenge for global health. In order to successfully combat SARS­CoV­2, the development of effective COVID­19 vaccines is crucial. In this context, recent studies have highlighted a high COVID­19 mortality rate in patients affected by ß­thalassemia, probably due to their co­existent immune deficiencies. In addition to a role in the severity of SARS­CoV­2 infection and in the mortality rate of COVID­19­infected patients with thalassemia, immunosuppression is expected to deeply affect the effectivity of anti­COVID­19 vaccines. In the context of the interplay between thalassemia­associated immunosuppression and the effectiveness of COVID­19 vaccines, the employment of immunomodulatory molecules is hypothesized. For instance, short­term treatment with mammalian target of rapamycin inhibitors (such as everolimus and sirolimus) has been found to improve responses to influenza vaccination in adults, with benefits possibly persisting for a year following treatment. Recently, sirolimus has been considered for the therapy of hemoglobinopathies (including ß­thalassemia). Sirolimus induces the expression of fetal hemoglobin (and this may contribute to the amelioration of the clinical parameters of patients with ß­thalassemia) and induces autophagy (thereby reducing the excessive levels of α­globin). It may also finally contribute to the mobilization of erythroid cells from the bone marrow (thereby reducing anemia). In the present study, the authors present the hypothesis that sirolimus treatment, in addition to its beneficial effects on erythroid­related parameters, may play a crucial role in sustaining the effects of COVID­19 vaccination in patients with ß­thalassemia. This hypothesis is based on several publications demonstrating the effects of sirolimus treatment on the immune system.