Treatment of Erythroid Precursor Cells from β-Thalassemia Patients with Cinchona Alkaloids: Induction of Fetal Hemoglobin Production

β-thalassemias are among the most common inherited hemoglobinopathies worldwide and are the result of autosomal mutations in the gene encoding β-globin, causing an absence or low-level production of adult hemoglobin (HbA). Induction of fetal hemoglobin (HbF) is considered to be of key importance for the development of therapeutic protocols for β-thalassemia and novel HbF inducers need to be proposed for pre-clinical development. The main purpose on this study was to analyze Cinchona alkaloids (cinchonidine, quinidine and cinchonine) as natural HbF-inducing agents in human erythroid cells. The analytical methods employed were Reverse Transcription quantitative real-time PCR (RT-qPCR) (for quantification of γ-globin mRNA) and High Performance Liquid Chromatography (HPLC) (for analysis of the hemoglobin pattern). After an initial analysis using the K562 cell line as an experimental model system, showing induction of hemoglobin and γ-globin mRNA, we verified whether the two more active compounds, cinchonidine and quinidine, were able to induce HbF in erythroid progenitor cells isolated from β-thalassemia patients. The data obtained demonstrate that cinchonidine and quinidine are potent inducers of γ-globin mRNA and HbF in erythroid progenitor cells isolated from nine β-thalassemia patients. In addition, both compounds were found to synergize with the HbF inducer sirolimus for maximal production of HbF. The data obtained strongly indicate that these compounds deserve consideration in the development of pre-clinical approaches for therapeutic protocols of β-thalassemia.


Introduction
β-thalassemias are among the most common inherited hemoglobinopathies worldwide, and are the result of more than 300 autosomal mutations of the gene encoding β-globin, causing an absence or low-level synthesis of this protein (and consequently of adult hemoglobin, HbA) in erythropoietic cells [1][2][3][4][5]. The phenotypes range widely from asymptomatic (β-thalassemia trait or carrier) to clinically relevant anemia, which is categorized as transfusion-dependent β-thalassemia (TDT, including thalassemia major) and non-transfusion-dependent β-thalassemia (NTDT, thalassemia intermedia) [1]. Figure 1 shows that cinchonidine (CincD), quinidine (QuinD) and cinchonine (CincN) are all able to induce erythroid differentiation of human K562 cells in a concentrationdependent fashion. Determinations were performed after 5, 6 and 7 days of differentiation induction.  [50], lymphangioleiomyomatosis (LAM) [51], tuberous sclerosis complex [52] and different types of cancers [53][54][55]. Figure 1 shows that cinchonidine (CincD), quinidine (QuinD) and cinchonine (CincN) are all able to induce erythroid differentiation of human K562 cells in a concentration-dependent fashion. Determinations were performed after 5, 6 and 7 days of differentiation induction. After this induction period, cells were stained with benzidine in order to identify the hemoglobin-containing cells [26,[36][37][38]. These data confirm the already-reported effects of these Cinchona alkaloids on the K562 cell system [28]. The induction of K562 differentiation was found to be associated, as expected, with the inhibition of cell After this induction period, cells were stained with benzidine in order to identify the hemoglobin-containing cells [26,[36][37][38]. These data confirm the already-reported effects of these Cinchona alkaloids on the K562 cell system [28]. The induction of K562 differentiation was found to be associated, as expected, with the inhibition of cell proliferation, as reported in the K562 cell system for several other HbF inducers, such as hydroxyurea, mithramycin, rapamycin, cisplatin analogues and trimethylangelicin [25][26][27]37,[56][57][58][59]. This effect of cinchonidine, quinidine and cinchonine on K562 cell proliferation is presented and comparatively analyzed in Supplementary Figures S1 and S2. The induction of K562 erythroid differentiation by the studied Cinchona alkaloids was similar (with respect to the extent of the induced proportion of benzidine-positive cells) to that of sirolimus (rapamycin), despite the fact that the effects of sirolimus were appreciable at 100-200 nM (Supplementary Figure  S3). These differences among putative erythroid inducers were fully expected [60]. For example, butyrates are active at mM concentrations [61].

Cinchonidine, Quinidine and Cinchonine Potentiate Sirolimus-Induced Differentiation of K562 Erythroleukemia Cells
In order to verify possible combined effects of Cinchona alkaloids and sirolimus, we treated K562 cells simultaneously with different concentrations of these molecules. The obtained results of the treatment, reported in Figure 2, show that CincD, QuinD and CincN were able to further increase the erythroid differentiation activity of sirolimus, when this HbF inducer was used at 100 nM ( Figure 2, panels A, C and E) and 200 nM ( Figure 2, panels B, D and F).
In this experiment, K562 cells were cultured with increasing concentrations of cinchonidine, quinidine and cinchonine in the presence of 100 nM and 200 nM sirolimus, as indicated. The proportion of benzidine-positive K562 cells was determined after 5, 6 and 7 days of treatment. The data presented in Figure 2G are related to the use of suboptimal concentrations of CincD, QuinD and CincN (60, 40 and 50 µM, respectively) in the presence of 100 and 200 nM sirolimus. When the inducers (either sirolimus, or cinchonidine, quinidine and cinchonine) were used alone, the proportion of benzidine-positive hemoglobin-containing K562 cells was about 10-30%. On the contrary, when the inducers were used in combination, the proportion of benzidine-positive hemoglobin-containing K562 cells was always found to exceed 50% (the maximum level was reached using cinchonidine in combination with 200 nM sirolimus). The increased % of benzidine-positive cells compared with single administrations was always found to be statistically highly significant (p < 0.001). Untreated K562 displayed a proportion of benzidine-positive cells which never exceeded 2-5% (see, for instance, the microphotographs presented in the left part of Figure 3). Figure 3 shows a representative microscopic analysis of the benzidine assays performed under some of the different experimental conditions described in Figure 2. The data give clear evidence that, in addition to the already reported increase in the proportion of benzidine-positive cells, and additional feature of combined treatments was evident, i.e., that all the benzidine-positive cells were brightly stained with benzidine-hydrogen peroxide solution (black arrowheads).
On the contrary, the presence of slightly stained benzidine-positive cells was clearly appreciable in singularly treated K562 cells (white arrowheads). While the representative data shown in Figure 3 were obtained using CincD plus SIR combination, the data obtained using QuinD/SIR and CincN/SIR combinations were found to be very similar (as shown in Supplementary Figure S4). Although this assay was not quantitative, the results obtained sustain the concept that the combined treatments lead to the highest levels of increase in the proportion of brightly stained benzidine-positive K562 cells. In order to verify this hypothesis using a more quantitative assay, RT-qPCR was performed on isolated RNA.    Figure 4 shows that the erythroid differentiation induced by cinchonidine, q and cinchonine was associated with an increase in the production of α-globin 4A,C,E) and γ-globin mRNAs ( Figure 4B,D,F). The analysis of the expression of and γ-globin mRNAs was performed by RT-qPCR.

The Effects of Cinchonidine, Quinidine and Cinchonine on K562 Erythroid Differen Are Associated with a Modulation of Expression of α-Globin and γ-Globin Genes
Interestingly, when used singularly, the increases in the production of both induced by CincD ( Figure 4A,B), QuinD ( Figure 4C,D), and CincN ( Figure 4E found to be similar to those found when 100 nM and 200 nM sirolimus was emp > 0.05). In addition, the increased levels of γ-globin mRNAs were similar to those o described by Iftikhar et al. [28]. On the contrary, when cinchonidine, quinidine chonine were used in combination with 100 nM and 200 nM sirolimus, a sharp in the content of α-globin mRNA (p < 0.01) and a less extensive but still significant increase in γ-globin mRNAs were found, fully in agreement with the effects of th pounds on sirolimus-induced K562 erythroid differentiation (Figures 2 and 3). The effects of CincD, QuinD, and CincN were dose-dependent, but clearly even when sub-optimal concentrations of compounds were employed.
Among the different combinations studied, those based on cinchonidine an dine were found to be the most effective in terms of inducing increases in the pr of benzidine-positive cells and increased expression of globin genes (Supplemen ures S5 and S6). Therefore, these two compounds were selected for further stud erythroid precursor cells (ErPCs) from β-thalassemia patients as an experiment system.   shows that the erythroid differentiation induced by cinchonidine, quinidine and cinchonine was associated with an increase in the production of α-globin ( Figure 4A,C,E) and γ-globin mRNAs ( Figure 4B,D,F). The analysis of the expression of α-globin and γ-globin mRNAs was performed by RT-qPCR.
Interestingly, when used singularly, the increases in the production of both mRNAs induced by CincD ( Figure 4A,B), QuinD ( Figure 4C,D), and CincN ( Figure 4E,F) were found to be similar to those found when 100 nM and 200 nM sirolimus was employed (p > 0.05). In addition, the increased levels of γ-globin mRNAs were similar to those originally described by Iftikhar et al. [28]. On the contrary, when cinchonidine, quinidine and cinchonine were used in combination with 100 nM and 200 nM sirolimus, a sharp increase in the content of α-globin mRNA (p < 0.01) and a less extensive but still significant (p < 0.05) increase in γ-globin mRNAs were found, fully in agreement with the effects of these compounds on sirolimus-induced K562 erythroid differentiation (Figures 2 and 3). In Figure 4, relevant examples of the p values obtained are shown, while the complete statistical analysis is presented in Supplementary Figures S5-S7.
The effects of CincD, QuinD, and CincN were dose-dependent, but clearly evident even when sub-optimal concentrations of compounds were employed.
Among the different combinations studied, those based on cinchonidine and quinidine were found to be the most effective in terms of inducing increases in the proportion of benzidine-positive cells and increased expression of globin genes (Supplementary Figures S5 and S6). Therefore, these two compounds were selected for further studies using erythroid precursor cells (ErPCs) from β-thalassemia patients as an experimental model system.

Cinchonidine and Quinidine Induce HbF and γ-Globin mRNA in Erythroid Precursor Cells (ErPCs) from β-Thalassemia Patients
Patients were recruited at the Thalassemia Centre of Azienda Ospedaliera-

Cinchonidine and Quinidine Induce HbF and γ-Globin mRNA in Erythroid Precursor Cells (ErPCs) from β-Thalassemia Patients
Patients were recruited at the Thalassemia Centre of Azienda Ospedaliera-Universitaria S. Anna (Ferrara, Italy). In total, 10 patients were enrolled. Informed written consent from all participants was obtained before recruiting them into the study. Different genotypes were present in the recruited cohort: five patients were β 0 39/β + IVSI-110, two patients were β + IVSI-110/β + IVSI-110 and three patients were β 0 39/β 0 39. ErPCs were isolated from the β-thalassemia patients and cultured, as described elsewhere [59], with erythropoietin in the presence of sirolimus, cinchonidine and quinidine administered alone (this was performed in ErPC cultures from all the ten patients) or in combination (this was performed in ErPC cultures from five patients).
In Figure 5, the HPLC profiles of two representative ErPC populations are shown, one isolated from patient #10 ( Figure 5A-C) and the other from patient #5 ( Figure 5D-F), exhibiting a differential response to cinchonidine ( Figure 5B,E) and quinidine ( Figure 5C,F) treatment.
The ErPCs from patient #10 exhibited a 69.57% and 118.04% increase in HbF after treatment with cinchonidine and quinidine, respectively (see the raw data shown in Supplementary Table S1). A lower increase in HbF was obtained when the ErPCs from patient #1 were employed (in this case, the HbF increase was 5.40% and 9.53% for cinchonidine and quinidine, respectively). patients were β + IVSI-110/β + IVSI-110 and three patients were β 0 39/β 0 39. ErPCs were is lated from the β-thalassemia patients and cultured, as described elsewhere [59], wi erythropoietin in the presence of sirolimus, cinchonidine and quinidine administere alone (this was performed in ErPC cultures from all the ten patients) or in combinatio (this was performed in ErPC cultures from five patients).
In Figure 5, The ErPCs from patient #10 exhibited a 69.57% and 118.04% increase in HbF aft treatment with cinchonidine and quinidine, respectively (see the raw data shown in Sup plementary Table S1). A lower increase in HbF was obtained when the ErPCs from patien  Figure 6 shows the effects of cinchonidine and quinidine on the ErPCs from all the 10 recruited β-thalassemia patients. All the raw data are reported in Supplementary  Table S1. As clearly evident, increases in the proportion of HbF (% of all the accumulated hemoglobins) were found in the treated ErPCs from most of the recruited β-thalassemia patients ( Figure 6A).
In fact, the HbF increase was significant in the treated cells when the obtained values were compared to those found in untreated cells ( Figure 6B).
As expected for HbF inducers, the differences were not statistically significant when the data of CincD-and/or QuinD-treated ErPCs were compared with those obtained using SIR or HU.
The HbF increase was, as expected, associated with increase in γ-globin mRNA (Figure 6C). Moreover, in this case, the increases in γ-globin mRNA (2.26 ± 0.37 and 2.04 ± 0.36 folds for cinchonidine and quinidine, respectively) were similar to the increases found in sirolimus-treated (2.21 ± 0.29) and hydroxyurea-treated (2.41 ± 0.35) ErPCs. The expected slight inhibitory effects of CincD and QuinD on ErPC cell proliferation were similar to those of the validated HbF inducers SIR and HU (Supplementary Figure S8).  Figure 7 shows the data obtained when ErPCs from five patients were treated, in addition to the treatments already mentioned in Section 2.4, with cinchonidine and quinidine in the presence of 100 nM sirolimus. The data reported are related to increase in the % of HbF and of γ-globin mRNA content. As clearly evident, the ErPC cultures exhibiting the highest levels of % of HbF and increased γ-globin mRNA are those treated with the two alkaloids and sirolimus (the raw data are reported in Supplementary Table S2). For instance, the % increase in HbF was 66.30 ± 25.09 and 82.30 ± 37.18 in ErPCs co-treated with sirolimus plus cinchonidine or sirolimus plus quinidine, respectively. These values were higher than those found when single drugs were added (20.45 ± 13.82, 31.61 ± 11.62 and 54.83 ± 19.52 for sirolimus, cinchonidine and quinidine, respectively). When the values relative to the treatments with CincD plus SIR and with QuinD plus SIR were compared to the treatment with the reference HbF inducers HU and SIR, the differences in the % of HbF incresae were found to be highly significant ( Figure 7A, upper part of the panel). On the contrary, when the values of CincD or QuinD are compared to HU or SIR, the differences were not significant (p > 0.2).  Figure 6B presented this increase in the % of HbF with respect to untreated ErPCs and gives evidence for an HbF increase comparable to that of sirolimus and the established HbF inducer hydroxyurea.

Cinchonidine and Quinidine Potentiate Sirolimus-Mediated Induction of HbF and γ-Globin mRNA in ErPCs from β-Thalassemia Patients
In fact, the HbF increase was significant in the treated cells when the obtained values were compared to those found in untreated cells ( Figure 6B).
As expected for HbF inducers, the differences were not statistically significant when the data of CincD-and/or QuinD-treated ErPCs were compared with those obtained using SIR or HU.
The HbF increase was, as expected, associated with increase in γ-globin mRNA ( Figure 6C). Moreover, in this case, the increases in γ-globin mRNA (2.26 ± 0.37 and 2.04 ± 0.36 folds for cinchonidine and quinidine, respectively) were similar to the increases found in sirolimus-treated (2.21 ± 0.29) and hydroxyurea-treated (2.41 ± 0.35) ErPCs. The expected slight inhibitory effects of CincD and QuinD on ErPC cell proliferation were similar to those of the validated HbF inducers SIR and HU (Supplementary Figure S8). Figure 7 shows the data obtained when ErPCs from five patients were treated, in addition to the treatments already mentioned in Section 2.4, with cinchonidine and quinidine in the presence of 100 nM sirolimus. The data reported are related to increase in the % of HbF and of γ-globin mRNA content. As clearly evident, the ErPC cultures exhibiting the highest levels of % of HbF and increased γ-globin mRNA are those treated with the two alkaloids and sirolimus (the raw data are reported in Supplementary Table S2). For instance, the % increase in HbF was 66.30 ± 25.09 and 82.30 ± 37.18 in ErPCs co-treated with sirolimus plus cinchonidine or sirolimus plus quinidine, respectively. These values were higher than those found when single drugs were added (20.45 ± 13.82, 31.61 ± 11.62 and 54.83 ± 19.52 for sirolimus, cinchonidine and quinidine, respectively). When the values relative to the treatments with CincD plus SIR and with QuinD plus SIR were compared to the treatment with the reference HbF inducers HU and SIR, the differences in the % of HbF incresae were found to be highly significant ( Figure 7A, upper part of the panel). On the contrary, when the values of CincD or QuinD are compared to HU or SIR, the differences were not significant (p > 0.2).

Treatment of ErPCs from β-Thalassemia Patients with Cinchonidine and Qu Associated with a Sharp Decrease in the Free α-Globin Chains
Reduction in the excess α-globin should be considered as an import the development of therapeutic interventions of β-thalassemia, since the decreases the lifespan of the red-blood cells, causes ineffective erythropoie jor determinant of the clinical severity of β-thalassemia [42].
The effects of cinchonidine and quinidine on the free α-globin chain the ErPCs from the recruited β-thalassemia patients displaying α-globin >

Treatment of ErPCs from β-Thalassemia Patients with Cinchonidine and Quinidine Is Associated with a Sharp Decrease in the Free α-Globin Chains
Reduction in the excess α-globin should be considered as an important objective in the development of therapeutic interventions of β-thalassemia, since the excess α-globin decreases the lifespan of the red-blood cells, causes ineffective erythropoiesis and is a major determinant of the clinical severity of β-thalassemia [42].
The effects of cinchonidine and quinidine on the free α-globin chains produced by the ErPCs from the recruited β-thalassemia patients displaying α-globin > 2.5 are summarized in Figure 8. All the raw data are reported in Supplementary Table S3.

Discussion
Induction of fetal hemoglobin (HbF) is considered a very promising strategy in the therapy of β-thalassemia and sickle-cell disease (SCD). The gene-therapy-mediated induction of γ-globin gene expression and HbF production in erythroid cells was described. In addition, approaches using genome editing are available in forms that are finalized to the induction of HbF following the elimination of genomic sequences encoding for transcriptional repressors or genomic sequences targeted by these regulatory factors [15][16][17][18][19].
While some positive results have been described based on HbF induction by gene therapy and gene editing, the safety of these approaches are still to be determined. In addition, it is expected that the costs of these therapeutic interventions will be very high [21][22][23].
Accordingly, the validation of the clinical relevance of already-known HbF inducers and the characterization of novel HbF inducers are still projects of great interest [24][25][26][27]. In this context, a paper was recently published by Iftikhar et al. on the effects of Cinchona alkaloids (cinchonidine and quinidine) as natural fetal hemoglobin-inducing agents in human erythroleukemia cells [28]. This study was very interesting, despite the fact that the key results were obtained using only K562 cells as an in vitro experimental model system.
The key conclusions of our study are the following: (a) cinchonidine and quinidine are inducers of an increase in the % of HbF in erythroid progenitor cells isolated from βthalassemia patients; (b) cinchonidine and quinidine potentiate the activity of sirolimus (a HbF inducer employed in clinical trials). These data sustain the concept that cinchonidine and quinidine should be considered for further studies aimed at developing protocols for the treatment of β-thalassemia patients. Our data show that the HbF induction efficiency of cinchonidine and quinidine is similar to that of hydroxyurea (the reference HbF inducer) and sirolimus ( Figure 6).
In addition to the induction of changes in HbF expression, cinchonidine and quinidine might also act through a reduction in the excess free α-globins present in the erythroid cells of β-thalassemia patients. This reduction should be considered a key objective in the use of molecules for therapeutic interventions in the management of β-thalassemia, since the excess α-globin is one of key factors causing short lifespans of the red- As clearly evident, a decrease in the % of the free α-globin peak was found in the treated ErPCs from most of the recruited β-thalassemia patients (see Supplementary Table  S3). Of great interest is a lower reduction in the free α-globin chains when ErPCs were treated with sirolimus or hydroxyurea. An average α-globin peak reduction of 27.98% was found with sirolimus, while the % average reduction with hydroxyurea was only 10.78%. Higher and more significant reductions (p = 0.024504 and 0.006129, respectively) were found with cinchonidine and quinidine (49.15% and 58.79%, respectively). These data suggest that, in addition to increased expression of γ-globin genes and HbF production, cinchonidine and quinidine might exert their beneficial effects on ErPCs through a decrease in the excess free α-globin chains.

Discussion
Induction of fetal hemoglobin (HbF) is considered a very promising strategy in the therapy of β-thalassemia and sickle-cell disease (SCD). The gene-therapy-mediated induction of γ-globin gene expression and HbF production in erythroid cells was described. In addition, approaches using genome editing are available in forms that are finalized to the induction of HbF following the elimination of genomic sequences encoding for transcriptional repressors or genomic sequences targeted by these regulatory factors [15][16][17][18][19].
While some positive results have been described based on HbF induction by gene therapy and gene editing, the safety of these approaches are still to be determined. In addition, it is expected that the costs of these therapeutic interventions will be very high [21][22][23].
Accordingly, the validation of the clinical relevance of already-known HbF inducers and the characterization of novel HbF inducers are still projects of great interest [24][25][26][27]. In this context, a paper was recently published by Iftikhar et al. on the effects of Cinchona alkaloids (cinchonidine and quinidine) as natural fetal hemoglobin-inducing agents in human erythroleukemia cells [28]. This study was very interesting, despite the fact that the key results were obtained using only K562 cells as an in vitro experimental model system.
The key conclusions of our study are the following: (a) cinchonidine and quinidine are inducers of an increase in the % of HbF in erythroid progenitor cells isolated from β-thalassemia patients; (b) cinchonidine and quinidine potentiate the activity of sirolimus (a HbF inducer employed in clinical trials). These data sustain the concept that cinchonidine and quinidine should be considered for further studies aimed at developing protocols for the treatment of β-thalassemia patients. Our data show that the HbF induction efficiency of cinchonidine and quinidine is similar to that of hydroxyurea (the reference HbF inducer) and sirolimus ( Figure 6).
In addition to the induction of changes in HbF expression, cinchonidine and quinidine might also act through a reduction in the excess free α-globins present in the erythroid cells of β-thalassemia patients. This reduction should be considered a key objective in the use of molecules for therapeutic interventions in the management of β-thalassemia, since the excess α-globin is one of key factors causing short lifespans of the red-blood cells with associated ineffective erythropoiesis [42]. Interestingly, this therapeutic relevant target is reached very efficiently using cinchonidine and quinidine, as both are more efficient in reducing the excess free α-globin than hydroxyurea and sirolimus ( Figure 8). Further studies will clarify whether the reduction in the excess α-globin is associated with the activation of autophagy, as proposed elsewhere [42].
In terms of combined treatments, we decided to employ sirolimus [35] as this HbF inducer is, at present, employed in two ongoing clinical trials: NCT03877809 (A Personalized Medicine Approach for β-thalassemia Transfusion Dependent Patients: Testing sirolimus in a First Pilot Clinical Trial) and NCT04247750 (Treatment of β-thalassemia Patients with Rapamycin: From Pre-clinical Research to a Clinical Trial) [45]. Further studies will verify whether the Cinchona alkaloids employed in this study potentiate the activity of other HbF inducers, including hydroxyurea, that are extensively employed in the treatment of β-thalassemia and sickle-cell disease [62,63]. One of the limits of our study is that the mechanism(s) of action was not experimentally evaluated. Further studies are required to understand this specific issue in our ErPC model system. However, published studies support the hypothesis that the mechanism(s) of action of Cinchona alkaloids and sirolimus are sharply different. In fact, Cinchona alkaloids are reported to inhibit cytochrome P450 enzyme 2D6 and the transport protein P-glycoprotein [64,65], while sirolimus is firmly established as an mTOR inhibitor [35,66].
The data obtained in our study strongly support the concept that cinchonidine and quinidine might be employed in combination with sirolimus in order to maximize its effects on in vivo-treated β-thalassemia patients.
In this respect, it is interesting to observe that cinchonidine and quinidine might be more active than hydroxyurea, which is one of the most important reference compounds when clinical treatment of β-thalassemia and sickle-cell disease is considered [62,63]. Further studies employing analyses of the effects on transcriptome and proteome, as well as confirming the presence of increased HbF within selected cell populations, are needed in order to verify whether the increase in the % of HbF reported in the present study is accompanied by a clinically relevant increase in the content of HbF in each treated erythroid cell.
Moreover, in order to propose a possible protocol for therapeutic purposes, a proofof-principle showing in vivo effects on animal model systems is highly recommended, as well as a careful analysis of the relationship between the effects on HbF and the presence of DNA polymorphisms associated with the predisposition of patients to high HbF induction.

Patients Recruitment
Cultures of erythroid progenitors were derived from the peripheral blood of β-thalassemia patients. Patients were recruited at the Day Hospital Thalassemia and Hemoglobinophaties of Azienda Ospedaliera-Universitaria S. Anna (Ferrara, Italy). All the patients received a patient information sheet to read and time to clarify doubts with investigators before consenting. All the participants signed an informed consent form on the basis of approvals of the Ethical Committee in charge of human studies at the University Hospital. The recruited patients were all transfusion dependent and not under hydroxyurea therapy. Treatments were performed on cultured ErPCs derived from patients blood isolated just before transfusion.

Chemical Reagents for Cell Culture Treatments
The reagents used for K562 treatments (rapamycin (sirolimus, SIR), cat. R0395; hydroxyurea (HU), cat. H8627; cinchonine (CincN), cat. 27370; cinchonidine (CincD), cat. C80407; quinidine (QuinD), cat. 22600) were purchased from Sigma Aldrich (St. Louis, MO, USA). HU was solubilized in sterile deionized H 2 O, whereas rapamycin, cinchonine, cinchonidine and quinidine were solubilized in ethanol and stored at −20 • C. Stock solution of rapamycin was prepared at 5 mM and 20 mM for each of the Cinchona alkaloids used. We used a concentration of sirolimus known to induce both K562 and erythroid precursor cells from β-thalassemia patients [37]. These stocks were further diluted to the indicated concentrations in culture medium prior to experimentation. All the treatments were performed by adding the compounds once at the beginning of the culturing period.

In Vitro Culture of Erythroid Progenitors from β-Thalassemia Patients
The two-phase liquid culture procedure was employed as previously described [37,59]. Mononuclear cells were isolated from peripheral blood samples of β-thalassemia patients: 20-25 mL of peripheral blood were collected before transfusion from patients who gave informed consent. A mixture of blood and PBS 1× at a 1:1 ratio was stratified on top of Lympholyte ® -H Cell Separation Media (Cedarlane, Burlington, NC, USA). After isolation, the mononuclear cell layer was washed three times by adding 1× PBS solution and seeded in α-minimal essential medium

Reverse Transcription and Quantitative Real-Time PCR (RT-qPCR)
For gene expression analysis, 500 ng of total RNA were reverse transcribed using the TaqMan ® Reverse Transcription Reagents kit and random hexamers (Applied Biosystems, Foster City, CA, USA). The RT-qPCR assay was carried out using gene-specific double fluorescently labeled probes. The reaction mixture had a final volume of 25 µL and was composed of Prime Time ® Gene Expression Master Mix 1× (IDT, Tema Research, Castenaso, BO, Italy), the pairs of forward and reverse primers (α, β, γ, together or GAPDH, RPL13A, ACTB together) used at 500 nM concentration and the probes (α, β, γ, together or GAPDH, RPL13A, ACTB together) used at 250 nM concentration. The probes that contained 6carboxyfluorescein (FAM) and hexachloro-6-carboxyfluorescein (HEX) as chromogenic molecules at 5 were quenched by the Iowa Black ® FQ molecule at 3 , while probes that contained indocarbocyanine (Cy5) were quenched by Iowa Black ® RQ. After an initial step for the denaturation at 95 • C for 2 min, the reactions were performed for 50 cycles consisting of two phases, 95 • C for 10 s and 60 • C for 45 s.

HPLC Analysis of Hemoglobins
To evaluate the effective quantity of the various types of hemoglobin produced by the cultured erythroid cells after treatment, High-Performance Liquid Chromatography was performed. The ErPCs were centrifuged at 2000 rpm for 6 minutes and washed with PBS (Phosphate buffered saline). The pellet was then resuspended in a predefined volume of water for HPLC (Sigma-Aldrich, St. Louis, MO, USA). This was followed by 3 freeze/thaw cycles on dry ice in order to lyse the cells and obtain the protein extracts. Lysates were centrifuged for 5 min at 14,000 rpm and the supernatant was collected. Hemoglobin analysis was performed by loading the protein extracts into a PolyCAT-A cation exchange column, and they were then eluted in a sodium-chloride-BisTris-KCN aqueous mobile phase using the HPLC Beckman Coulter Instrument System Gold 126 Solvent Module-166 Detector, which allowed us to obtain a quantification of the hemoglobins present in the sample. The reading was performed at a wavelength of 415nm, and a commercial solution of purified human HbAF (Sigma-Aldrich) extracts was used as a standard. The values thus obtained were processed using "32 Karat software".

Statistical Analysis
All the data were normally distributed and presented as mean ± S.E.M. Statistical differences between groups were compared using a paired t-test or a one-way repeated measures ANOVA (ANalyses of VAriance between groups) followed by LSD post-hoc tests. Statistical differences were considered significant when p < 0.05 (*) and highly significant when p < 0.01 (**).
Funding: This study was sponsored by the Wellcome Trust (Innovator Award 208872/Z/17/Z) and AIFA (AIFA-2016-02364887). The research leading to these results has also received funding by the UE THALAMOSS Project (Thalassemia Modular Stratification System for Personalized Therapy of Beta-Thalassemia; no. 306201-FP7-HEALTH-2012-INNOVATION-1) and FIR and FAR funds from the University of Ferrara. This research was also supported by ALT (Associazione per la lotta alla Talassemia "Rino Vullo"-Ferrara, AVLT (Associazione Veneta per la Lotta alla Talassemia "Elio Zago" -APS), and by the Interuniversity Consortium for Biotechnology (C.I.B.), Italy. C.Z. was supported by a fellowship from "Tutti per Chiara Onlus".

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki and the use of human material was approved by the Ethics Committee of Ferrara's District, protocol name: THAL-THER, document number 533/2018/Sper/AOUFe, approved on 14 November 2018. All samples of peripheral blood were obtained after receiving written informed consent from donor patients or their legal representatives.
Informed Consent Statement: Informed consent for the testing of HbF inducers in erythroid progenitors derived from peripheral blood was obtained from all β-thalassemia patients involved in the study before the blood was drawn.
Data Availability Statement: Most of the raw data are included in Supplementary Materials. Additional information will be freely available upon request to the correspondence authors.