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.

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

Introduction: ß-thalassemia is caused by autosomal mutations in the ß-globin gene, which induce the absence or low-level synthesis of ß-globin in erythroid cells. It is widely accepted that a high production of fetal hemoglobin (HbF) is beneficial for patients with ß-thalassemia. Sirolimus, also known as rapamycin, is a lipophilic macrolide isolated from a strain of Streptomyces hygroscopicus that serves as a strong HbF inducer in vitro and in vivo. In this study, we report biochemical, molecular, and clinical results of a sirolimus-based NCT03877809 clinical trial (a personalized medicine approach for ß-thalassemia transfusion-dependent patients: testing sirolimus in a first pilot clinical trial, Sirthalaclin). Methods: Accumulation of γ-globin mRNA was analyzed using reverse-transcription quantitative polymerase chain reaction (PCR), while the hemoglobin pattern was analyzed using high-performance liquid chromatography (HPLC). The immunophenotype was analyzed using a fluorescence-activated cell sorter (FACS), with antibodies against CD3, CD4, CD8, CD14, CD19, CD25 (for analysis of peripheral blood mononuclear cells), or CD71 and CD235a (for analysis of in vitro cultured erythroid precursors). Results: The results were obtained in eight patients with the ß+/ß+ and ß+/ß0 genotypes, who were treated with a starting dosage of 1 mg/day sirolimus for 24-48 weeks. The first finding of this study was that the expression of γ-globin mRNA increased in the blood and erythroid precursor cells isolated from ß-thalassemia patients treated with low-dose sirolimus. This trial also led to the important finding that sirolimus influences erythropoiesis and reduces biochemical markers associated with ineffective erythropoiesis (excess free α-globin chains, bilirubin, soluble transferrin receptor, and ferritin). A decrease in the transfusion demand index was observed in most (7/8) of the patients. The drug was well tolerated, with minor effects on the immunophenotype, and an only side effect of frequently occurring stomatitis. Conclusion: The data obtained indicate that low doses of sirolimus modify hematopoiesis and induce increased expression of γ-globin genes in a subset of patients with ß-thalassemia. Further clinical trials are warranted, possibly including testing of the drug in patients with less severe forms of the disease and exploring combination therapies.

Impact of the preparation method of red cell concentrates on transfusion indices in thalassemia patients: A randomized crossover clinical trial.

BACKGROUND: The average hemoglobin content of red cell concentrates (RCC) varies depending on the method of preparation. Surprisingly less data are available concerning the clinical impact of those differences. STUDY DESIGN AND METHODS: The effects of two types of RCC (RCC-A, RCC-B) on transfusion regime were compared in a non-blinded, prospective, randomized, two-period, and crossover clinical trial. RCC-A was obtained by whole blood leukoreduction and subsequent plasma removal, RCC-B removing plasma and buffy coat first, followed by leukoreduction. Eligible patients were adult, with transfusion-dependent thalassemia (TDT). RESULTS: RCC-A contained 63.9 (60.3-67.8) grams of hemoglobin per unit (median with 1st and 3rd quartile), RCC-B 54.5 (51.0-58.2) g/unit. Fifty-one patients completed the study. With RCC-B, the average pre-transfusion hemoglobin concentration was 9.3 ± 0.5 g/dl (mean ± SD), the average transfusion interval 14.2 (13.7-16.3) days, the number of RCC units transfused per year 39.3 (35.4-47.3), and the transfusion power index (a composite index) 258 ± 49. With RCC-A, the average pre-transfusion hemoglobin concentration was 9.6 ± 0.5 g/dl (+2.7%, effect size 0.792), the average transfusion interval 14.8 (14.0-18.5) days (+4.1%, effect size 0.800), the number of RCC units transfused per year 34.8 (32.1-42.5) (-11.4%, effect size -1.609), and the transfusion power index 272 ± 61 (+14.1%, effect size 0.997). All differences were statistically highly significant (p < .00001). The frequency of transfusion reactions was 0.59% with RCC-A and 0.56% with RCC-B (p = 1.000). CONCLUSION: To reduce the number of RCC units consumed per year and the number of transfusion episodes, TDT patients should receive RCC with the highest average hemoglobin content.