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BACKGROUND. The human bone marrow (BM) niche contains a population of mesenchymal stromal cells (MSCs) that provide physical support and regulate hematopoietic stem cell (HSC) homeostasis. β-Thalassemia (BT) is a hereditary disorder characterized by altered hemoglobin beta-chain synthesis amenable to allogeneic HSC transplantation and HSC gene therapy. Iron overload (IO) is a common complication in BT patients affecting several organs. However, data on the BM stromal compartment are scarce. METHODS. MSCs were isolated and characterized from BM aspirates of healthy donors (HDs) and BT patients. The state of IO was assessed and correlated with the presence of primitive MSCs in vitro and in vivo. Hematopoietic supportive capacity of MSCs was evaluated by transwell migration assay and 2D coculture of MSCs with human CD34 + HSCs. In vivo, the ability of MSCs to facilitate HSC engraftment was tested in a xenogenic transplant model, whereas the capacity to sustain human hematopoiesis was evaluated in humanized ossicle models. RESULTS. We report that, despite iron chelation, BT BM contains high levels of iron and ferritin, indicative of iron accumulation in the BM niche. We found a pauperization of the most primitive MSC pool caused by increased ROS production in vitro which impaired MSC stemness properties. We confirmed a reduced frequency of primitive MSCs in vivo in BT patients. We also discovered a weakened antioxidative response and diminished expression of BM niche–associated genes in BT-MSCs. This caused a functional impairment in MSC hematopoietic supportive capacity in vitro and in cotransplantation models. In addition, BT-MSCs failed to form a proper BM niche in humanized ossicle models. CONCLUSION. Our results suggest an impairment in the mesenchymal compartment of BT BM niche and highlight the need for novel strategies to target the niche to reduce IO and oxidative stress before transplantation. FUNDING. This work was supported by the SR-TIGET Core grant from Fondazione Telethon and by Ricerca Corrente.

Hematopoietic stem cells (HSCs) are regulated by signals from the bone marrow (BM) niche, which tune hematopoiesis at steady state and in hematologic disorders. To understand the HSC-niche interactions in altered non-malignant homeostasis, we elected as a paradigm β-thalassemia, a hemoglobin disorder. In this severe congenital anemia, secondary alterations to the primary hemoglobin defect have a potential impact on HSC-niche crosstalk. Here we report that HSCs in thalassemic mice (th3) have an impaired function, caused by the interaction with an altered BM niche. The HSC self-renewal defect is rescued upon transplantation into a normal microenvironment, thus proving the active role of BM stroma. Consistently with the common finding of osteoporosis in patients, we found reduced bone deposition with decreased levels of parathyroid hormone (PTH), which is a key regulator of bone metabolism but also of HSC activity. In vivo activation of PTH signaling through the reestablished Jagged1 and osteopontin levels correlates with the rescue of the functional pool of th3 HSCs by correcting HSC-niche crosstalk. Reduced HSC quiescence is confirmed in thalassemic patients, along with altered features of the BM stromal niche. Our findings uncover a defect of HSCs in β-thalassemia induced by an altered BM microenvironment and provide new relevant insight for improving transplantation and gene therapy approaches.

Plerixafor and G-CSF combination mobilizes hematopoietic stem and progenitors cells with a distinct transcriptional profile and a reduced in vivo homing capacity compared to plerixafor alone For decades, bone marrow (BM) has been the preferred source of hematopoietic stem and progenitor cells (HSPCs) for transplants following myeloablative conditioning. At present, mobilized peripheral blood stem cells are commonly used for transplantation, particularly in the autologous setting.

Gene therapy clinical trials require rigorous non-clinical studies in the most relevant models to assess the benefit-to-risk ratio. To support the clinical development of gene therapy for β-thalassemia, we performed in vitro and in vivo studies for prediction of safety. First we developed newly GLOBE-derived vectors that were tested for their transcriptional activity and potential interference with the expression of surrounding genes. Because these vectors did not show significant advantages, GLOBE lentiviral vector (LV) was elected for further safety characterization. To support the use of hematopoietic stem cells (HSCs) transduced by GLOBE LV for the treatment of β-thalassemia, we conducted toxicology, tumorigenicity, and biodistribution studies in compliance with the OECD Principles of Good Laboratory Practice. We demonstrated a lack of toxicity and tumorigenic potential associated with GLOBE LV-transduced cells. Vector integration site (IS) studies demonstrated that both murine and human transduced HSCs retain self-renewal capacity and generate new blood cell progeny in the absence of clonal dominance. Moreover, IS analysis showed an absence of enrichment in cancer-related genes, and the genes targeted by GLOBE LV in human HSCs are well known sites of integration, as seen in other lentiviral gene therapy trials, and have not been associated with clonal expansion. Taken together, these integrated studies provide safety data supporting the clinical application of GLOBE-mediated gene therapy for β-thalassemia.

Hematopoietic stem cells (HSC) are regulated by signals from the bone marrow (BM) niche and little is known about their fate in altered hematological conditions associated to non-malignant diseases. In β-thalassemia ineffective erythropoiesis and secondary alterations, as abnormal regulation of bone metabolism, iron overload and hormonal factors, induce changes in the BM homeostasis with a potential impact on HSC-niche interaction. We addressed these unexplored issues in the murine disease model and in patients' cells. We investigated hematopoiesis in thalassemic Hbbth3/+ (th3) mutant mice and we found lower frequency, reduced quiescence and reconstituting potential of HSC. th3 HSC have impaired self-renewal, which is rescued upon transplantation in a normal BM, proving an active role of the niche microenvironment. Both stromal and hematopoietic components of the BM niche are altered in th3 mice. Consistently with the common finding of osteoporosis in patients, we found reduced bone deposition with decreased levels of parathyroid hormone (PTH), which is a key regulator of bone metabolism but also of HSC activity. Low PTH negatively affects bone deposition and expression of the Notch-ligand Jag1 by th3 mesenchymal and osteolineage cells, thus reducing the activation of Notch1 in HSC and consequently impairing their function. In vivo activation of PTH signaling through the reestablished Jag1-Notch1 pathway restores the functional pool of th3 HSC by correcting HSC-niche crosstalk. In addition to the stromal component of the BM, hematopoietic cells with a key role in regulating the fate of HSC, such as megakaryocytes (Mk), were found defective in maturation, possibly due to reduced circulating levels of thrombopoietin (TPO). We are currently investigating the molecular causes of dysmegakaryopoiesis and the Mk-HSC interaction in thalassemic mice. Strikingly, reduced HSC quiescence was confirmed in samples from patients affected by β-thalassemia, along with impaired stromal niche and megakaryopoiesis, thus highlighting the clinical relevance of our findings. Further investigation will unravel the multiple molecular mechanisms that affect in trans HSC functions in the complexity of the stressed thalassemic BM microenvironment. Our results uncover a defect of HSC in β-thalassemia, induced by an altered BM niche and provide new relevant insight for improving transplantation and gene therapy approaches. Disclosures No relevant conflicts of interest to declare.

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The bone marrow (BM) niche regulation and interactions with hematopoietic stem cells (HSC) have been extensively studied in steady state conditions and malignancies, but are still underexplored in hematological inherited disorders. We provided the first demonstration of impaired HSC function caused by an altered BM niche in a non-malignant disease, beta-thalassemia (BT) (Aprile et al., Blood 2020). BT is a globally widespread congenital hemoglobin disorder, resulting in severe anemia, ineffective erythropoiesis and multi-organ secondary complications, including bone alterations. Correction of the genetic defect is achieved by transplantation of HSC from healthy donors or autologous HSC from patients upon gene therapy. Since the quality and the engraftment of HSC depend on the BM microenvironment, niche-HSC crosstalk plays a crucial role for transplantation outcome. During the analysis of different components of the niche, we found reduced bone density in BT th3 mice, along with a defective HSC supporting activity by the BM stromal niche. Interestingly, osteoporosis is a constant hallmark in BT patients. We investigated the mechanisms underlying bone and HSC niche defects focusing on the role of fibroblast growth factor-23 (FGF-23), a hormone mainly secreted by osteocytes, but also by erythroid cells, which negatively modulates bone metabolism. Since FGF-23 is stimulated by the anemia-related factor erythropoietin (EPO), we hypothesized that the high EPO levels in BT might contribute to increase FGF-23, potentially affecting bone and BM niche homeostasis. We found high levels of circulating FGF-23 in th3 mice (wt vs. th3: 290.5±27.3 vs. 1823±136.1 pg/ml, p<0.0001) and in BT patients (HD vs. THAL: 94.7±1.8 vs. 117.2±5.3 RU/ml, p<0.01), associated to its increased expression by bone and BM erythroid cells. In vivo neutralization of EPO axis was sufficient to normalize FGF-23 levels (th3 vs. th3+anti-EPO: 1591±162.2 vs. 496.1±33.3 pg/ml, p<0.001), thus demonstrating the causative role of EPO. EPO stimulation and signaling inhibition strategies highlighted the involvement of Erk1/2 and Stat5 pathways in activating Fgf-23 transcription in bone and BM erythroid cells, respectively. To provide proof of concept data on the contribution of FGF-23 to BT bone and stromal niche alterations, we inhibited FGF-23 signaling. In vivo administration of FGF-23 blocking peptide rescued the trabecular bone density in th3 mice (th3 vs. th3+FGF23inh: 138.2±4.9 vs. 166.9±5.2 mg/cm 3, p<0.01). Short-term inhibition treatment (38 hours) was sufficient to enhance bone mineralization by acting on Alkaline Phosphatase and on the expression of the main regulators of mineralization Dmp1 and Mepe by osteocyte, whereas long-term administration (12 days) restored also osteoblast number and bone deposition. Importantly, FGF-23 inhibition normalized the expression of key niche molecules, such as Jagged-1 and osteopontin, involved in the functional crosstalk between HSC and the stromal niche. Consistently, the treatment restored the frequency of th3 HSC by expanding the pool of quiescent cells (th3 vs. th3+FGF23inh: 0.026 vs. 0.045% on Lin neg BM cells, p<0.01). FGF-23 inhibition had also a positive anti-apoptotic effect on the expanded BM erythroid compartment (th3 vs. th3+FGF23inh: 61.6±1.3 vs. 51.1±2.1% of BM Ter119 + cells, p<0.001), promoting the maturation of early erythroid precursors (th3 vs. th3+FGF23inh: 8.5±1 vs. 16.4±1.1% of BM Pro-Erythroblasts, p<0.0001), as already shown in models of secondary anemias. Evidence in BT patients showed negative correlations between FGF-23 levels and markers of bone homeostasis (e.g. osteocalcin R 2=0.88, p<0.05) and positive correlations with makers of ineffective erythropoiesis (e.g. circulating reticulocytes R 2=0.83, p<0.05), thus positioning FGF-23 as the molecule at the crossroads of erythropoiesis and bone metabolism in BT. Our findings uncover an underexplored role of FGF-23 in bone and BM niche defects in a primary anemia, as a condition of chronic EPO stimulation, and propose FGF-23 as the missing link between hematopoiesis and bone regulation. The inhibition of FGF-23 signaling might provide a novel strategy to ameliorate bone compartment and restore HSC-BM niche interactions in BT by a 'two birds with one stone' approach, with a potential translational relevance in improving HSC transplantation and gene therapy. Disclosures Cappellini: Celgene: Consultancy, Research Funding; Vifor: Consultancy; La Jolla: Research Funding; Protagonist Therapeutics: Research Funding; IONIS Pharmaceuticals: Consultancy; CRISPR Therapeutics: Research Funding; Novartis: Consultancy, Honoraria, Research Funding.