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

MicroRNA miR-93-5p regulates expression of IL-8 and VEGF in neuroblastoma SK-N-AS cells

The role of the microRNA miR-93-5p on the secretome profile and the expression levels of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) was investigated in the neuroblastoma SK-N-AS cell line by Bio-Plex analysis and RT-qPCR. The results indicate that VEGF and IL-8 are the major miR-93-5p molecular targets. This conclusion was based on in vitro transfection with pre-miR-93-5p and anti-miR-93-5p; these treatments inversely modulated both VEGF and IL-8 gene expression and protein release in the neuroblastoma SK-N-AS cell line. Computational analysis showed the presence of miR-93-5p consensus sequences in the 3'UTR region of both VEGF and IL-8 mRNAs, predicting possible interaction with miR-93-5p and confirming a potential regulatory role of this microRNA

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

Orphan Drugs and Potential Novel Approaches for Therapies of β-Thalassemia: Current Status and Future Expectations

Introduction: The β-thalassemias are rare diseases caused by more than 300 different mutations of the adult β-globin genes: they are treated with blood transfusions, chelation therapy or bone marrow transplantation.Areas Covered: This article reviews orphan drugs, related patent applications and related clinical trials on β-thalassemia, including novel therapeutic approaches and repurposing of orphan drugs.Expert Opinion: Patient stratification is expected to be applied for personalized therapeutic interventions for β-thalassemia patients. This will cover primary mutations of the β-globin gene and DNA polymorphisms that predict severity of the disease and response to therapy. Improved understanding of the regulation of γ-globin gene expression and the optimization of novel approaches for gene editing are expected to introduce innovative orphan drugs and/or approaches for fetal hemoglobin induction and gene correction, respectively. The possible combination between gene therapy and cellular therapy, including gene-correction, presents a new opportunity. A dramatic increase in the need for cellular biobanking (including also cord blood biobanking) is expected, given the possibility to intervene on patients-derived cells with bone marrow transplantation and/or generated induced pluripotent stem cells. The repurposing of drugs used in clinical trials for different pathologies is an interesting approach to providing novel drugs at low cost

Molecular Methods for Validation of the Biological Activity of Peptide Nucleic Acids Targeting MicroRNAsmiRNA Maturation

The involvement of microRNAs in human pathologies is a firmly established fact. Accordingly, the pharmacological modulation of their activity appears to be a very appealing issue in the development of new types of drugs (miRNA therapeutics). One of the most interesting issues is the possible development of miRNA therapeutics for development of anti-cancer molecules. In this respect appealing molecules are based on peptide nucleic acids (PNAs), displaying a pseudo-peptide backbone composed of N-(2-aminoethyl) glycine units and found to be excellent candidates for antisense and antigene therapies. The major limit in the use of PNAs for alteration of gene expression is the low uptake by eukaryotic cells. The aim of this chapter is to describe methods for determining the activity of PNAs designed to oncomiRNA targets, using as model system miR-221 and its target p27(Kipl) mRNA. The effects of PNAs targeting miR-221 are here presented discussing data obtained using as model system the human breast cancer cell line MDA-MB-231, in which miR-221 is up-regulated and p27(Kipl) down-regulated

Erythroid induction of K562 cells treated with mithramycin is associated with inhibition of raptor gene transcription and mammalian target of rapamycin complex 1 (mTORC1) functions

Rapamycin, an inhibitor of mTOR activity, is a potent inducer of erythroid differentiation and fetal hemoglobin production in β-thalassemic patients. Mithramycin (MTH) was studied to see if this inducer of K562 differentiation also operates through inhibition of mTOR. We can conclude from the study that the mTOR pathway is among the major transcript classes affected by mithramycin-treatment in K562 cells and a sharp decrease of raptor protein production and p70S6 kinase is detectable in mithramycin treated K562 cells. The promoter sequence of the raptor gene contains several Sp1 binding sites which may explain its mechanism of action. We hypothesize that the G+C-selective DNA-binding drug mithramycin is able to interact with these sequences and to inhibit the binding of Sp1 to the raptor promoter due to the following results: (a) MTH strongly inhibits the interactions between Sp1 and Sp1-binding sites of the raptor promoter (studied by electrophoretic mobility shift assays, EMSA); (b) MTH strongly reduces the recruitment of Sp1 transcription factor to the raptor promoter in intact K562 cells (studied by chromatin immunoprecipitation experiments, ChIP); (c) Sp1 decoy oligonucleotides are able to specifically inhibit raptor mRNA accumulation in K562 cells. In conclusion, raptor gene expression is involved in mithramycin-mediated induction of erythroid differentiation of K562 cells and one of its mechanism of action is the inhibition of Sp1 binding to the raptor promoter

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 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