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

Cristina Zuccato, Lucia Carmela Cosenza, Matteo Zurlo, Jessica Gasparello, Chiara Papi, Elisabetta D'Aversa, Giulia Breveglieri, Ilaria Lampronti, Alessia Finotti, Monica Borgatti, Chiara Scapoli, Alice Stievano, Monica Fortini, Eric Ramazzotti, Nicola Marchetti, Marco Prosdocimi, Maria Rita Gamberini, Roberto Gambari, Cristina Zuccato, Lucia Carmela Cosenza, Matteo Zurlo, Jessica Gasparello, Chiara Papi, Elisabetta D'Aversa, Giulia Breveglieri, Ilaria Lampronti, Alessia Finotti, Monica Borgatti, Chiara Scapoli, Alice Stievano, Monica Fortini, Eric Ramazzotti, Nicola Marchetti, Marco Prosdocimi, Maria Rita Gamberini, Roberto Gambari

Abstract

Introduction: β-thalassemia is caused by autosomal mutations in the β-globin gene, which induce the absence or low-level synthesis of β-globin in erythroid cells. It is widely accepted that a high production of fetal hemoglobin (HbF) is beneficial for patients with β-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 β-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 β+/β+ and β+/β0 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 β-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 β-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.

Keywords: fetal hemoglobin; ineffective erythropoiesis; sirolimus; transfusion requirement; β-thalassemia; γ-globin mRNA.

Conflict of interest statement

Conflict of interest statement: The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: M.P. is the administrator of ‘Rare Partners srl Impresa Sociale’ to whom the rights for the patent WO 2004/004697 on the use of sirolimus in β-thalassemia have been assigned.

© The Author(s), 2022.

Figures

Figure 1.
Figure 1.
Flowchart summarizing the NCT03877809 Sirthalaclin clinical trial and the key analysis presented in this study. Five patients concluded the trial at V8 (180 days), as indicated. HbF and excess of free α-globin chains were analyzed using HPLC. Content of γ-globin mRNA was analyzed using RT-qPCR. The scheme of the trial has been described elsewhere.
Figure 2.
Figure 2.
Representative HPLC patterns obtained after exposure of ErPCs isolated from patients (a) n.9, (b) n.14, (c) n.15, and (d) n.17 to sirolimus. The arrows indicate the positions of the HbA, HbA2, and HbF0 peaks.
Figure 3.
Figure 3.
Blood levels of sirolimus (ng/mL). For calculating the average values, the samples with sirolimus blood concentrations p-value for the comparison of V8 data to V3 data, samples from patient n.24 were not considered.
Figure 4.
Figure 4.
Increase in the content of γ-globin mRNA in the blood of patients treated with sirolimus (a–c). Representative data obtained from the blood samples of patient n.11 using (a) RPL13A, (b) GAPDH, and (c) β-actin control sequences, as indicated. (d) Increase in the content of γ-globin mRNA in the blood. The data represent fold values with respect to V2 of patient n.11. (e) Average increase values of γ-globin mRNA expressed as fold content with respect to V2 of patient n.11. Raw data are presented in Supplementary Table S5. N = 8 (V2–V8) and 3 (V11).
Figure 5.
Figure 5.
Increase in the content of γ-globin mRNA in the ErPCs isolated from patients treated with sirolimus. (a) Increase in the content of γ-globin mRNA in the ErPCs. The data represent fold values with respect to V2 of patient n.24. (b) Average increase in the content of γ-globin mRNA. The data represent fold values with respect to V2 of patient n.24. Raw data are presented in Supplementary Table S6. N = 8 (V2–V8) and 3 (V11).
Figure 6.
Figure 6.
Increase in percentage of HbF in EPO-induced ErPCs isolated at V6. (a) Representative HPLC analysis of EPO-treated ErPCs from patients n.11 and n.18 at V2 and V6 Sirthalaclin visits. The arrows indicate positions of the HbA, HbA2, and HbF peaks. (b) Changes in the percentage of HbF at V6 (black histograms) with respect to V2 (white histograms). The cumulative data from all the analyzed patients are also shown.
Figure 7.
Figure 7.
(a) Changes in free α-globin chains, (b) bilirubin, (c) sTFR, and (d) ferritin levels, following treatment with sirolimus. Only six patients were analyzed for changes of free α-globin chains, since two patients exhibited lack of detectable α-globin peak on HPLC analysis. Raw data related to panels B–D are presented in Supplementary Tables S7–S9. N = 8 (V2–V8) and 3 (V11).
Figure 8.
Figure 8.
Changes in the transfusion demand (TD) when data at V2 and V8 are considered (p = 0.006). N = 8.
Figure 9.
Figure 9.
Immunophenotype of PBMCs from sirolimus-treated β-thalassemia patients: FACS analysis. A representative example of the gating strategy employed is given in Supplementary Figure S5A–C. The summary of the results obtained is shown focusing on the following markers associated with the various lymphocyte subpopulations: (a) CD14, (b) CD19, (c) CD3, (d) CD4, (e) CD8, and (f) CD25. With this panel of antibodies, we were able to subdivide the PBMCs population into monocytes (CD14+), B cells (CD14−/CD3−/CD19+), T cells (CD14−/CD3+), CD4+ T cells (CD14−/CD3+/CD4+), CD8+ T cells (CD14−/CD3+/CD8+), and CD8+ activated T cells (CD14−/CD3+/CD8+/CD25+). Average changes of the immunophenotype pattern at V2, V6, V8, and V11 are reported. N = 8 (V2–V8) and 3 (V11).

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