Efficacy of ruxolitinib as inducer of fetal hemoglobin in primary erythroid cultures from sickle cell and beta-thalassemia patients

High levels of HbF may ameliorate the clinical course of β-thalassaemia and SCD. Hydroxyurea (HU) is the only HbF inducer approved for the treatment of patients. However not all patients respond to the treatment, for this reason it is noteworthy to identify new HbF inducers. Ruxolitinib is a JAK inhibitor that decreases the phosphorilation of STAT proteins. In particular STAT3 is a repressor of gamma-globin gene. The decrease of STAT3 phosphorilation could derepress gamma-globin gene and reactivate its trascription. In this study we evaluated the efficacy of ruxolitinib as inducer of HbF production. The analyses were performed in cultured erythroid progenitors from 16 beta-thalassemia intermedia (TI) and 4 sickle cell disease (SCD) patients. The use of quantitative RT-PCR technique allowed us to determine the increase of gamma-globin mRNA expression in human erythroid cultured cells treated with ruxolitinib. The results of our study demonstrated an increase in vitro of gamma-globin mRNA expression in almost all patients. These data suggest that ruxolitinib could be a good candidate to be used in vivo for the treatment of hemoglobinopathies

321. Sea Urchin sns Chromatin Insulator Prevents Silencing and Positional Effect Variegation of Oncoretroviral Vectors Transgene Expression in Murine Erythroid Cell Line

Silencing and position effect are considered significant obstacles to obtain a consistent level of transgene expression in viral gene therapy. Furthermore recent studies had shown that retroviruses tend to land on active genes with the potential consequence of insertional mutagenesis. The inclusion of elements, such as chromatin insulators, capable to insulate a gene from the surrounding chromatin effects at the integration site should improve both efficacy and safety of gene therapy vectors. We have previously characterized a 265 bp insulator element, termed sns, localized at the 3' end of the early histone H2A gene of the sea urchin Paracentrotus lividus. This sequence contains three cis-acting elements (Box A, Box B, and Box C+T) all needed for the enhancer blocking activity in both sea urchin and human cells. By colony assays, in human (K562) and mouse (Mel) erythroid cell lines, we have recently demonstrated that the sns insulator displays directional enhancer-blocking activity in that it interferes with the communication between the human beta-globin enhancer (LCR) and the gamma-globin promoter. By electrophoretic mobility shift assays (EMSA) we found bindings of sns insulator with the erythroid specific GATA1 and the ubiquitous Oct1, and Sp1 transcription factors

Study on Hydroxyurea Response in Hemoglobinopathies Patients Using Genetic Markers and Liquid Erythroid Cultures

Increased expression of fetal hemoglobin (HbF) may ameliorate the clinical course of hemoglobinopathies. Hydroxyurea (HU) is the only inducer approved for the treatment of these diseases able to stimulate HbF production but patients’ response is highly variable indicating the utility of the identification of pharmacogenomic biomarkers in order to predict pharmacological treatment efficacy. To date few studies to evaluate the role of genetic determinants in HU response have been conducted showing contradictory results. In this study we analyzed BCL11A, GATA-1, KLF-1 genes and γ-globin promoter in 60 alleles from 30 hemoglobinopathies patients under HU treatment to assess the role of these markers in HU response. We did not find any association between these genetic determinants and HU response. Before treatment started, the same patients were analyzed in vitro using liquid erythroid cultures in a test able to predict their response to HU. The results of our analysis confirm the absence of pharmacogenomic biomarker associated to HU response indicating that, the quantification of γ-globin mRNA fold increase remains the only method able to predict in vivo patients response to the drug

2-Pentadecyl-2-Oxazoline, the Oxazoline of Pea, Modulates Carrageenan-Induced Acute Inflammation

N-acylethanolamines (NAEs) involve a family of lipid molecules existent in animal and plant, with N-palmitoylethanolamide (PEA) that arouses great attention owing to its anti-inflammatory, analgesic and neuroprotective activities. Because PEA is produced on demand and exerts pleiotropic effects, the modulation of specific amidases for NAEs (and in particular NAE-hydrolyzing acid amidase NAAA, which is more selective for PEA) could be a condition to preserve its levels. Here we investigate the effect of 2-Pentadecyl-2-oxazoline (PEA-OXA) the oxazoline of PEA, on human recombinant NAAA in vitro and in an established model of Carrageenan (CAR)-induced rat paw inflammation. PEA-OXA dose-dependently significantly inhibited recombinant NAAA and, orally administered to rats (10 mg/kg), limiting histological damage, thermal hyperalgesia and the increase of infiltrating inflammatory cells after CAR injection in the rat right hindpaw, compared to ultramicronized PEA given orally at the same dose (10 mg/kg). These effects were accompanied by elevation of paw PEA levels. Moreover, PEA-OXA markedly reduced neutrophil infiltration and pro-inflammatory cytokine release and prevented CAR-induced IκB-α degradation, nuclear translocation of NF-κB p65, the increase of inducible nitric oxide synthase, cyclooxygenase-2, intercellular adhesion molecule-1, and mast cell activation. Experiments in PPAR-α knockout mice showed that the anti-inflammatory effects of PEA-OXA were not dependent on the presence of PPAR-α receptors. In conclusion, NAAA modulators as PEA-OXA could help to maximize the tissue availability of PEA by increasing its levels and anti-inflammatory effects

Oral Ultramicronized Palmitoylethanolamide: Plasma and Tissue Levels and Spinal Anti-hyperalgesic Effect

Palmitoylethanolamide (PEA) is a pleiotropic lipid mediator with established anti-inflammatory and anti-hyperalgesic activity. Ultramicronized PEA (PEA-um) has superior oral efficacy compared to naïve (non-micronized) PEA. The aim of the present study was two-fold: (1) to evaluate whether oral PEA-um has greater absorbability compared to naïve PEA, and its ability to reach peripheral and central tissues under healthy and local inflammatory conditions (carrageenan paw edema); (2) to better characterize the molecular pathways involved in PEA-um action, particularly at the spinal level. Rats were dosed with 30 mg/kg of [13C]4-PEA-um or naïve [13C]4-PEA by oral gavage, and [13C]4-PEA levels quantified, as a function of time, by liquid chromatography/atmospheric pressure chemical ionization/mass spectrometry. Overall plasma levels were higher in both healthy and carrageenan-injected rats administered [13C]4-PEA-um as compared to those receiving naïve [13C]4-PEA, indicating the greater absorbability of PEA-um. Furthermore, carrageenan injection markedly favored an increase in levels of [13C]4-PEA in plasma, paw and spinal cord. Oral treatment of carrageenan-injected rats with PEA-um (10 mg/kg) confirmed beneficial peripheral effects on paw inflammation, thermal hyperalgesia and tissue damage. Notably, PEA-um down-regulated distinct spinal inflammatory and oxidative pathways. These last findings instruct on spinal mechanisms involved in the anti-hyperalgesic effect of PEA-um in inflammatory pain

The Sea Urchin sns5 Insulator Protects Retroviral Vectors From Chromosomal Position Effects by Maintaining Active Chromatin Structure

Silencing and position-effect (PE) variegation (PEV), which is due to integration of viral vectors in heterochromatin regions, are considered significant obstacles to obtaining a consistent level of transgene expression in gene therapy. The inclusion of chromatin insulators into vectors has been proposed to counteract this position-dependent variegation of transgene expression. Here, we show that the sea urchin chromatin insulator, sns5, protects a recombinant γ-retroviral vector from the negative influence of chromatin in erythroid milieu. This element increases the probability of vector expression at different chromosomal integration sites, which reduces both silencing and PEV. By chromatin immunoprecipitation (ChIP) analysis, we demonstrated the specific binding of GATA1 and OCT1 transcription factors and the enrichment of hyperacetylated nucleosomes to sns5 sequences. The results suggest that this new insulator is able to maintain a euchromatin state inside the provirus locus with mechanisms that are common to other characterized insulators. On the basis of its ability to function as barrier element in erythroid milieu and to bind the erythroid specific factor GATA1, the inclusion of sns5 insulator in viral vectors may be of practical benefit in gene transfer applications and, in particular, for gene therapy of erythroid disorders