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

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

Nonsense mutations generate in-frame stop codons in mRNA leading to a premature arrest of translation. Functional consequences of premature termination codons (PTCs) include the synthesis of truncated proteins with loss of protein function causing severe inherited or acquired diseases. A therapeutic approach has been recently developed that is based on the use of chemical agents with the ability to suppress PTCs (read-through) restoring the synthesis of a functional full-length protein. Research interest for compounds able to induce read-through requires an efficient high throughput large scale screening system. We present a rapid, sensitive and quantitative method based on a dual-fluorescence reporter expressed in the yeast Saccharomyces cerevisiae to monitor and quantitate read-through at PTCs. We have shown that our novel system works equally well in detecting read-through at all three PTCs UGA, UAG and UAA. [ABSTRACT FROM AUTHOR]

Abstract: The PKI55 protein was identified in our laboratory as specific protein kinase C inhibitor. We previously demonstrated that PKI55 is poorly translated in vivo and acts promoting PKC degradation and establishing a feedback loop of inhibition. However, our understanding of mechanisms by which the expression of PKI55 is regulated, is limited. In the present work we investigated the mRNA expression of PKI55 in human tissues by Northern blotting and RT-PCR, demonstrating that it is highly expressed in brain tissue. Moreover, since the computational analysis of the gene promoter region showed two sites (Box 1 and Box 2) similar to consensus sequences for AP1 and GAGA factors, we investigated their ability to bind to these proteins. Electrophoretic mobility shift assays showed that GAGA factors preferentially interacted with Box 2, while AP1 elements linked preferentially Box 1 sequence. We suggest that the interaction of these transcription factors with Box 1 and Box 2 could regulate the transcription of the PKI55 gene and, consequently, the expression of PKC. [Copyright &y& Elsevier]

The human AβH-J-J locus is a genomic sequence which generates three functionally distinct proteins, the enzyme aspartyl-β-hydroxylase (AβH), the structural protein of sarcoplasmic reticulum junctin, and the membrane-bound calcium binding protein junctate. The first and second exons are mutually exclusive when mature mRNAs are produced. Moreover, the use of different splice donors has been shown to be involved in the generation of protein diversity by alternative splicing. As to transcriptional regulation, two promoters (P1 and P2) were identified. When the P1 and P2 promoter sequences are compared, important differences are clearly detectable. The most interesting result emerging from studies focused on the P2 promoter is that the calcium-dependent transcriptional factor MEF-2 activates the transcription of junctin, junctate, and AβH in excitable tissues and, to a lesser extent, in kidney. No Sp1 binding sites are present in the P2 promoter. In contrast, P1 promoter contains GC-rich sequences, which have homologies with the Sp1 consensus binding site. [ABSTRACT FROM AUTHOR]

We studied the effects of rapamycin on cultures of erythroid progenitors derived from the peripheral blood of 10 β-thalassaemia patients differing widely with respect to their potential to produce foetal haemoglobin (HbF). For this, we employed the two-phase liquid culture procedure for growing erythroid progenitors, high performance liquid chromatography for analysis of HbF production and reverse transcription polymerase chain reaction for quantification of the accumulation of globin mRNAs. The results demonstrated that rapamycin induced an increase of HbF in cultures from all the β-thalassaemia patients studied and an increase of their overall Hb content/cell. The inducing effect of rapamycin was restricted to γ-globin mRNA accumulation, being only minor for β-globin and none for α-globin mRNAs. The ability of rapamycin to preferentially increase γ-globin mRNA content and production of HbF in erythroid precursor cells from β-thalassaemia patients is of great importance as this agent (also known as sirolimus or rapamune) is already in clinical use as an anti-rejection agent following kidney transplantation. These data suggest that rapamycin warrants further evaluation as a potential therapeutic drug in β-thalassaemia and sickle cell anaemia. [ABSTRACT FROM AUTHOR]

Peptide nucleic acids (PNAs) have been demonstrated to be very useful tools for gene regulation at different levels and with different mechanisms of action. In the last few years the use of PNAs for targeting microRNAs (anti-miRNA PNAs) has provided impressive advancements. In particular, targeting of microRNAs involved in the repression of the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is defective in cystic fibrosis (CF), is a key step in the development of new types of treatment protocols. In addition to the anti-miRNA therapeutic strategy, inhibition of miRNA functions can be reached by masking the miRNA binding sites present within the 3′UTR region of the target mRNAs. The objective of this study was to design a PNA masking the binding site of the microRNA miR-145-5p present within the 3′UTR of the CFTR mRNA and to determine its activity in inhibiting miR-145-5p function, with particular focus on the expression of both CFTR mRNA and CFTR protein in Calu-3 cells. The results obtained support the concept that the PNA masking the miR-145-5p binding site of the CFTR mRNA is able to interfere with miR-145-5p biological functions, leading to both an increase of CFTR mRNA and CFTR protein content. [ABSTRACT FROM AUTHOR]

Background: Nonsense mutations promote premature translational termination, introducing stop codons within the coding region of mRNAs and causing inherited diseases, including thalassemia. For instance, in β039 thalassemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, ribosomal read-through molecules, such as aminoglycoside antibiotics, have been tested on mRNAs carrying premature stop codons. These findings have introduced new hopes for the development of a pharmacological approach to the β039 thalassemia therapy. While several strategies, designed to enhance translational read-through, have been reported to inhibit NMD efficiency concomitantly, experimental tools for systematic analysis of mammalian NMD inhibition by translational read-through are lacking. Results: We developed a human cellular model of the β039 thalassemia mutation with UPF-1 suppressed and showing a partial NMD suppression. Conclusions: This novel cellular model could be used for the screening of molecules exhibiting preferential read-through activity allowing a great rescue of the mutated transcripts. [ABSTRACT FROM AUTHOR]

Since the identification of microRNAs (miRNAs) involved in the regulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, miRNAs known to down-regulate the expression of the CFTR and associated proteins have been investigated as potential therapeutic targets. Here we show that miR-101-3p, targeting the 3′-UTR sequence of the CFTR mRNA, can be selectively inhibited by a peptide nucleic acid (PNA) carrying a full complementary sequence. With respect to clinical relevance of microRNA targeting, it is expected that reduction in concentration of miRNAs (the anti-miRNA approach) could be associated with increasing amounts of target mRNAs. Consistently to this hypothesis, we report that PNA-mediated inhibition of miR-101-3p was accompanied by CFTR up-regulation. Next Generation Sequencing (NGS) was performed in order to verify the effects of the anti-miR-101-3p PNA on the Calu-3 miRNome. Upon inhibition of miR-101-3p we observed a fold change (FC) expression <2 of the majority of miRNAs (403/479, 84.13%), whereas we identified a list of dysregulated miRNAs, suggesting that specific miRNA inhibition (in our case miR-101-3p) might be accompanied by alteration of expression of other miRNAs, some of them known to be involved in Cystic Fibrosis (CF), such as miR-155-5p and miR-125b-5p. Image 1 • microRNAs are deeply involved in the regulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. • miR-101-3p targets the 3′-UTR sequence of the CFTR mRNA. • In Calu-3 cells miR-101-3p can be selectively inhibited by a fully complementary peptide nucleic acid (PNA). • PNA-mediated inhibition of miR-101-3p was accompanied by CFTR up-regulation. • The effects of the anti-miR-101-3p PNA on the Calu-3 miRNome are accompanied by alteration of miRNAs involved in Cystic Fibrosis. [ABSTRACT FROM AUTHOR]