Expression of γ-globin genes in β-thalassemia patients treated with sirolimus: results from a pilot clinical trial (Sirthalaclin)

Abstract

Introduction: The β-thalassemias are due to autosomal mutations of the β-globin gene, inducing absence or low-level synthesis of β-globin in erythroid cells. It is widely accepted that high production of fetal hemoglobin (HbF) is beneficial for β-thalassemia patients. Sirolimus, also known as rapamycin, is a lipophilic macrolide isolated from a strain of Streptomyces hygroscopicus found to be a strong HbF inducer in vitro and in vivo. In this study, we report biochemical, molecular and clinical results of the sirolimus-based NCT03877809 clinical trial (A Personalized Medicine Approach for β-thalassemia Transfusion Dependent Patients: Testing sirolimus in a First Pilot Clinical Trial: Sirthalaclin). Methods: Accumulation of γ-globin mRNA was analyzed by reverse-transcription-quantitative PCR and the hemoglobin pattern by HPLC. The immunophenotype was analyzed by FACS using antibodies against CD3, CD4, CD8, CD14, CD19, CD25. Results: The results were obtained in 8 patients with β+/β+ and β+/β0 genotypes, treated with a starting dosage of 1 mg/day sirolimus for 24-48 weeks. The first finding of the study was that expression of γ-globin mRNA was increased in blood and erythroid precursor cells isolated from β-thalassemia patients treated with low-dose sirolimus. A second important conclusion of our trial was that sirolimus influences erythropoiesis and reduces biochemical markers associated to ineffective erythropoiesis (I.E.) (excess of free α-globin chains, bilirubin, soluble transferrin receptor and ferritin). In most (7/8) of the patients a decrease of the transfusion index was observed. The drug was well tolerated with minor effects on immunophenotype, the only side effect being frequently occurring stomatitis. Conclusions: The data obtained indicate that sirolimus given at low doses modifies hematopoiesis and induces increased expression of γ-globin genes in a sub-set of β-thalassemia patients. Further clinical trials are warranted, considering the possibility to test the drug in patients with less severe forms of the disease and exploring combination therapies.

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

“…D. Ratios of CD4 + /CD8 + T cells. A complete summary of the immunophenotype is reported in FigureS2, extending those reported by Zuccato et al40 List of antibodies employed for T memory Lymphocyte subset typing information on the following CD4 + (Figure3A) and CD8 + (Figure3B)subpopulations: naive, central memory (CM), transitional memory (TM) T cells, effector memory (EM) and effector memory expressing CD45RA (EMRA). These populations are of relevance, because identify T cells with different biological functions, and are clearly highlighted by antibodies staining.…”

“…Plasma samples were isolated from β-Thalassemia patients by direct centrifugation of the whole blood, as described in Zuccato et al 40 Proteins were measured using Bio-Plex Human Cytokine 27-plex Assay (Bio-Rad) as suggested by the manufacturer and described in Gasparello et al 53 and in the Supplementary Materials (SM1).…”

“…It is widely accepted that high production of foetal haemoglobin (HbF) is beneficial for β‐Thalassemia patients 35–38 . In a recent study, we have reported biochemical, molecular and clinical results of the Sirolimus‐based NCT03877809 clinical trial (A Personalized Medicine Approach for β‐Thalassemia Transfusion Dependent Patients: Testing Sirolimus in a First Pilot Clinical Trial: Sirthalaclin) 39,40 . The rationale of this trial was that Sirolimus is of interest in β‐Thalassemia, since it induces the expression of foetal haemoglobin (and this might contribute to ameliorate the clinical parameters of these patients), induces autophagy (thereby reducing the excess of free α‐globin) and, finally, might contribute to mobilization of erythroid cell from the bone marrow (thereby reducing anaemia).…”

“…suspended in 200 μl of PBS and analysed by flow cytometry using the BD FACSCanto II cell analyser (Becton Dickinson) as previously reported 40. …”

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

“…In most (7/8) of the patients a decrease of the transfusion demand index was indeed observed. The drug was well tolerated with minor effects on immunophenotype, the only side effect being frequently occurring stomatitis 65 .…”