Search

Sickle cell disease (SCD) is due to a mutation in the β-globin (HBB) gene causing the production of the toxic sickle hemoglobin (HbS, a2βS2). Transplantation of autologous hematopoietic stem/progenitor cells (HSPCs) transduced with lentiviral vectors (LVs) expressing an anti-sickling β-globin (βAS) is a promising treatment; however, it is only partially effective and patients still present elevated HbS levels. Here, we developed a bifunctional LV expressing βAS3-globin and an artificial microRNA (amiR) specifically downregulating βS-globin expression with the aim of reducing HbS levels and favoring βAS3 incorporation into Hb tetramers. Efficient transduction of SCD HSPC by the bifunctional LV led to a substantial decrease of βS-globin transcripts in HSPC-derived erythroid cells, a significant reduction of HbS+red cells and effective correction of the sickling phenotype, outperforming βAS gene addition andBCL11Agene silencing strategies. The bifunctional LV showed a standard integration profile and neither the HSPC viability, engraftment and multi-lineage differentiation nor the erythroid transcriptome and miRNAome were affected by the treatment, confirming the safety of this therapeutic strategy. In conclusion, the combination of gene addition and gene silencing strategies can improve the efficacy of current LV-based therapeutic approaches without increasing the mutagenic vector load, thus representing a novel treatment for SCD.

Haematopoietic stem and progenitor cell (HSPC) gene therapy has emerged as an effective treatment modality for monogenic disorders of the blood system such as primary immunodeficiencies and β-thalassaemia. Medicinal products based on autologous HSPCs corrected using lentiviral and gammaretroviral vectors have now been approved for clinical use, and the site-specific genome modification of HSPCs using gene editing techniques such as CRISPR-Cas9 has shown great clinical promise. Preclinical studies have shown engineered HSPCs could also be used to cross-correct non-haematopoietic cells in neurodegenerative metabolic diseases. Here, we review the most recent advances in HSPC gene therapy and discuss emerging strategies for using HSPC gene therapy for a range of diseases.

Rare hematopoietic stem and progenitor cell (HSPC) pools outside the bone marrow (BM) contribute to blood production in stress and disease but remain ill-defined. Although nonmobilized peripheral blood (PB) is routinely sampled for clinical management, the diagnosis and monitoring potential of PB HSPCs remain untapped, as no healthy PB HSPC baseline has been reported. Here we comprehensively delineate human extramedullary HSPC compartments comparing spleen, PB, and mobilized PB to BM using single-cell RNA-sequencing and/or functional assays. We uncovered HSPC features shared by extramedullary tissues and others unique to PB. First, in contrast to actively dividing BM HSPCs, we found no evidence of substantial ongoing hematopoiesis in extramedullary tissues at steady state but report increased splenic HSPC proliferative output during stress erythropoiesis. Second, extramedullary hematopoietic stem cells/multipotent progenitors (HSCs/MPPs) from spleen, PB, and mobilized PB share a common transcriptional signature and increased abundance of lineage-primed subsets compared with BM. Third, healthy PB HSPCs display a unique bias toward erythroid-megakaryocytic differentiation. At the HSC/MPP level, this is functionally imparted by a subset of phenotypic CD71+ HSCs/MPPs, exclusively producing erythrocytes and megakaryocytes, highly abundant in PB but rare in other adult tissues. Finally, the unique erythroid-megakaryocytic–skewing of PB is perturbed with age in essential thrombocythemia and β-thalassemia. Collectively, we identify extramedullary lineage-primed HSPC reservoirs that are nonproliferative in situ and report involvement of splenic HSPCs during demand-adapted hematopoiesis. Our data also establish aberrant composition and function of circulating HSPCs as potential clinical indicators of BM dysfunction.

beta-thalassemia is a genetic disorder caused by mutations in the beta-globin gene, and characterized by anemia, ineffective erythropoiesis and iron overload. Patients affected by the most severe transfusion-dependent form of the disease (TDT) require lifelong blood transfusions and iron chelation therapy, a symptomatic treatment associated with several complications. Other therapeutic opportunities are available, but none is fully effective and/or applicable to all patients, calling for the identification of novel strategies. Transferrin receptor 2 (TFR2) balances red blood cells production according to iron availability, being an activator of the iron-regulatory hormone hepcidin in the liver and a modulator of erythropoietin signaling in erythroid cells. Selective Tfr2 deletion in the BM improves anemia and iron-overload in non-TDT mice, both as a monotherapy and, even more strikingly, in combination with iron-restricting approaches. However, whether Tfr2 targeting might represent a therapeutic option for TDT has never been investigated so far. Here, we prove that BM Tfr2 deletion improves anemia, erythrocytes morphology and ineffective erythropoiesis in the Hbb(th1/th2) murine model of TDT. This effect is associated with a decrease in the expression of alpha-globin, which partially corrects the unbalance with beta-globin chains and limits the precipitation of misfolded hemoglobin, and with a decrease in the activation of unfolded protein response. Remarkably, BM Tfr2 deletion is also sufficient to avoid long-term blood transfusions required for survival of Hbb(th1/th2) animals, preventing mortality due to chronic anemia and reducing transfusion-associated complications, such as progressive iron-loading. Altogether, TFR2 targeting might represent a promising therapeutic option also for TDT.

β-Thalassemia (BT) is one of the most common genetic diseases worldwide and is caused by mutations affecting β-globin production. The only curative treatment is allogenic hematopoietic stem/progenitor cells (HSPCs) transplantation, an approach limited by compatible donor availability and immunological complications. Therefore, transplantation of autologous, genetically-modified HSPCs is an attractive therapeutic option. However, current gene therapy strategies based on the use of lentiviral vectors are not equally effective in all patients and CRISPR/Cas9 nuclease-based strategies raise safety concerns. Thus, base editing strategies aiming to correct the genetic defect in patients’ HSPCs could provide safe and effective treatment. Here, we developed a strategy to correct one of the most prevalent BT mutations (IVS1-110 [G>A]) using the SpRY-ABE8e base editor. RNA delivery of the base editing system was safe and led to ∼80% of gene correction in the HSPCs of patients with BT without causing dangerous double-strand DNA breaks. In HSPC-derived erythroid populations, this strategy was able to restore β-globin production and correct inefficient erythropoiesis typically observed in BT both in vitro and in vivo. In conclusion, this proof-of-concept study paves the way for the development of a safe and effective autologous gene therapy approach for BT.

In 2016, the European Hematology Association (EHA) published the EHA Roadmap for European Hematology Research1 aiming to highlight achievements in the diagnostics and treatment of blood disorders, and to better inform European policy makers and other stakeholders about the urgent clinical and scientific needs and priorities in the field of hematology. Each section was coordinated by 1 to 2 section editors who were leading international experts in the field. In the 5 years that have followed, advances in the field of hematology have been plentiful. As such, EHA is pleased to present an updated Research Roadmap, now including 11 sections, each of which will be published separately. The updated EHA Research Roadmap identifies the most urgent priorities in hematology research and clinical science, therefore supporting a more informed, focused, and ideally a more funded future for European hematology research. The 11 EHA Research Roadmap sections include Normal Hematopoiesis; Malignant Lymphoid Diseases; Malignant Myeloid Diseases; Anemias and Related Diseases; Platelet Disorders; Blood Coagulation and Hemostatic Disorders; Transfusion Medicine; Infections in Hematology; Hematopoietic Stem Cell Transplantation; CAR-T and Other Cellbased Immune Therapies; and Gene Therapy.

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.

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. Keywords: thalassemia, gene therapy, hematopoiesis, hematopoietic stem cell, preclinical model, safety, lentiviral vector, biodistribution, genotoxicity, integration site

Hematopoietic stem cell gene therapy has become a successful therapeutic strategy for some inherited genetic disorders. Pre-clinical toxicity studies performed to support the human clinical trials using viral-mediated gene transfer and autologous hematopoietic stem and progenitor cell (HSPC) transplantation are complex and the use of mouse models of human diseases makes interpretation of the results challenging. In addition, they rely on the use of conditioning agents that must induce enough myeloablation to allow engraftment of transduced and transplanted HSPC. Busulfan and total body irradiation (TBI) are the most commonly used conditioning regimens in the mouse. Lenticular degeneration and atrophy of reproductive organs are expected histopathological changes. Proliferative and nonproliferative lesions can be observed with different incidence and distribution across strains and mouse models of diseases. The occurrence of these lesions can interfere with the interpretation of pre-clinical toxicity and tumorigenicity studies performed to support the human clinical studies. As such, it is important to be aware of the background incidence of lesions induced by different conditioning regimens. We review the histopathology results from seven long-term studies, five using TBI and two using busulfan.

Nuclear receptor coactivator 4 (NCOA4) promotes ferritin degradation and Ncoa4-ko mice in a C57BL/6 background show microcytosis and mild anemia, aggravated by iron deficiency. To understand tissue-specific contributions of NCOA4-mediated ferritinophagy we explored the effect of Ncoa4 genetic ablation in the iron-rich Sv129/J strain. Increased body iron content protects these mice from anemia and, in basal conditions, Sv129/J Ncoa4-ko mice show only microcytosis; nevertheless, when fed a low-iron diet they develop a more severe anemia compared to that of wild-type animals. Reciprocal bone marrow (BM) transplantation from wild-type donors into Ncoa4-ko and from Ncoa4-ko into wild-type mice revealed that microcytosis and susceptibility to iron deficiency anemia depend on BM-derived cells. Reconstitution of erythropoiesis with normalization of red blood count and hemoglobin concentration occurred at the same rate in transplanted animals independently of the genotype. Importantly, NCOA4 loss did not affect terminal erythropoiesis in iron deficiency, both in total and specific BM Ncoa4-ko animals compared to controls. On the contrary, upon a low iron diet, spleen from wild-type animals with Ncoa4-ko BM displayed marked iron retention compared to (wild-type BM) controls, indicating defective macrophage iron release in the former. Thus, erythropoietin administration failed to mobilize iron from stores in Ncoa4-ko animals. Furthermore, Ncoa4 inactivation in thalassemic mice did not worsen the hematologic phenotype. Overall our data reveal a major role for NCOA4-mediated ferritinophagy in macrophages to favor iron release for erythropoiesis, especially in iron deficiency.

Studies on gender disparity in academia generate constructive discussions to promote equality. In a recently published study, AlShebli et al. 2020 analyzed the role of informal mentorship in supporting early-career scientists and how gender may shape scientific careers. Besides presenting methodological flaws, the study culminates in the authors' conclusion that mentoring quality is determined by the mentor's gender, suggesting that female protégés reap more benefits when mentored by males rather than equally-impactful females. Despite acknowledging that possible causal factors were not considered in their analyses, they attest that the success of female scientists' careers relies on opposite-gender mentorships in terms of publication and impact. Although the authors state that these findings add a new perspective to the policy debate on the best ways to elevate the women in science, their conclusions reinforce the traditional patriarchal, biased scientific structure that stimulates a poorly diversified hierarchical chain in STEM. Here we highlight the study's methodological weaknesses and major issues that must be addressed to avoid the perpetuation of gender disparity in science.

Haematopoietic stem and progenitor cell (HSPC) gene therapy has emerged as an effective treatment modality for monogenic disorders of the blood system such as primary immunodeficiencies and β-thalassaemia. Medicinal products based on autologous HSPCs corrected using lentiviral and gammaretroviral vectors have now been approved for clinical use, and the site-specific genome modification of HSPCs using gene editing techniques such as CRISPR-Cas9 has shown great clinical promise. Preclinical studies have shown engineered HSPCs could also be used to cross-correct non-haematopoietic cells in neurodegenerative metabolic diseases. Here, we review the most recent advances in HSPC gene therapy and discuss emerging strategies for using HSPC gene therapy for a range of diseases.

Hematopoietic stem cell gene therapy has become a successful therapeutic strategy for some inherited genetic disorders. Pre-clinical toxicity studies performed to support the human clinical trials using viral-mediated gene transfer and autologous hematopoietic stem and progenitor cell (HSPC) transplantation are complex and the use of mouse models of human diseases makes interpretation of the results challenging. In addition, they rely on the use of conditioning agents that must induce enough myeloablation to allow engraftment of transduced and transplanted HSPC. Busulfan and total body irradiation (TBI) are the most commonly used conditioning regimens in the mouse. Lenticular degeneration and atrophy of reproductive organs are expected histopathological changes. Proliferative and nonproliferative lesions can be observed with different incidence and distribution across strains and mouse models of diseases. The occurrence of these lesions can interfere with the interpretation of pre-clinical toxicity and tumorigenicity studies performed to support the human clinical studies. As such, it is important to be aware of the background incidence of lesions induced by different conditioning regimens. We review the histopathology results from seven long-term studies, five using TBI and two using busulfan.

β-Thalassemia intermedia is a disorder characterized by ineffective erythropoiesis (IE), anemia, splenomegaly, and systemic iron overload. Novel approaches are being explored based on the modulation of pathways that reduce iron absorption (ie, using hepcidin activators like Tmprss6-antisense oligonucleotides [ASOs]) or increase erythropoiesis (by erythropoietin [EPO] administration or modulating the ability of transferrin receptor 2 [Tfr2] to control red blood cell [RBC] synthesis). Targeting Tmprss6 messenger RNA by Tmprss6-ASO was proven to be effective in improving IE and splenomegaly by inducing iron restriction. However, we postulated that combinatorial strategies might be superior to single therapies. Here, we combined Tmprss6-ASO with EPO administration or removal of a single Tfr2 allele in the bone marrow of animals affected by β-thalassemia intermedia (Hbbth3/+). EPO administration alone or removal of a single Tfr2 allele increased hemoglobin levels and RBCs. However, EPO or Tfr2 single-allele deletion alone, respectively, exacerbated or did not improve splenomegaly in β-thalassemic mice. To overcome this issue, we postulated that some level of iron restriction (by targeting Tmprss6) would improve splenomegaly while preserving the beneficial effects on RBC production mediated by EPO or Tfr2 deletion. While administration of Tmprss6-ASO alone improved the anemia, the combination of Tmprss6-ASO + EPO or Tmprss6-ASO + Tfr2 single-allele deletion produced significantly higher hemoglobin levels and reduced splenomegaly. In conclusion, our results clearly indicate that these combinatorial approaches are superior to single treatments in ameliorating IE and anemia in β-thalassemia and could provide guidance to translate some of these approaches into viable therapies.

Hematopoietic stem cells (HSCs) are regulated by signals from the bone marrow (BM) niche that tune hematopoiesis at steady state and in hematologic disorders. To understand HSC-niche interactions in altered nonmalignant homeostasis, we selected β-thalassemia, a hemoglobin disorder, as a paradigm. In this severe congenital anemia, alterations secondary to the primary hemoglobin defect have a potential impact on HSC-niche cross talk. 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 after cell transplantation into a normal microenvironment, thus proving the active role of the BM stroma. Consistent 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 correlated with the rescue of the functional pool of th3 HSCs by correcting HSC-niche cross talk. Reduced HSC quiescence was confirmed in thalassemic patients, along with altered features of the BM stromal niche. Our findings reveal a defect in HSCs in β-thalassemia induced by an altered BM microenvironment and provide novel and relevant insight for improving transplantation and gene therapy approaches.

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 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 Evidence in BT patients showed negative correlations between FGF-23 levels and markers of bone homeostasis (e.g. osteocalcin R 2=0.88, p 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.

Background Transferrin Receptor 2 (TFR2) is a protein expressed in the liver and in the erythroid compartment. Hepatic TFR2 activates the transcription of hepcidin, the master regulator of iron homeostasis, and its inactivation causes iron overload. Erythroid TFR2 interacts with Erythropoietin (EPO) receptor and its deletion enhances erythropoiesis increasing EPO sensitivity of erythroid cells. We recently demonstrated that bone marrow (BM) Tfr2 loss improves anemia in a murine model of β-thalassemia. We hypothesized that the same approach might represent a therapeutic option also for anemias due to insufficient EPO production, as anemia of Chronic Kidney Disease (CKD). Indeed, anemia is a common complication of CKD, since EPO production is inhibited by progressive renal failure. In addition, chronic inflammation that parallels renal damage decreases EPO responsiveness of erythroid cells and enhances the production of hepcidin. Increased hepcidin levels limit iron absorption from the diet and release from the stores, reducing iron supply to erythropoiesis. All these factors contribute to anemia development. Replacement therapy with erythropoiesis stimulating agents (ESA), usually combined with iron supplementation, is effective but may lead to cardio-vascular side effects. Thus, novel and more specific strategies are needed. Aims Here, we exploit different murine models of selective Tfr2 inactivation to test whether Tfr2 targeting corrects anemia of CKD. In this context, BM Tfr2 inhibition is expected to stimulate erythropoiesis and the simultaneous downregulation of hepatic Tfr2 to correct iron deficiency. Methods We induced CKD using an adenine-rich diet in mice with: (1) BM-specific Tfr2 deletion (Tfr2 BMKO), generated through BM transplantation; (2) reduced Tfr2 hepatic expression, obtained treating wild-type mice with anti-Tfr2 antisense oligonucleotides (Tfr2-ASO); (3) germline Tfr2 inactivation in the whole organism (Tfr2KO). Results Renal damage was comparable among all the mice analyzed, excluding a differential effect of the diet in the various groups. Tfr2BMKO mice showed enhanced erythropoiesis relative to controls, due to the increased EPO responsiveness of erythroid cells lacking Tfr2, as suggested by the over-activation of the EPO-EPOR signaling pathway despite comparable EPO levels. Tfr2BMKO mice maintained higher red blood cell (RBC) count than controls for the entire protocol. Hemoglobin (Hb) levels, higher in Tfr2BMKO mice for 6 weeks, reached levels of controls at 8 weeks, concomitant with relative hypoferremia. These results indicate that BM Tfr2 deletion transiently prevents anemia until iron availability is adequate to the enhanced erythropoiesis. Then we investigated the potential beneficial effect of increasing iron availability through Tfr2-ASOs treatment, which efficiently decreased hepatic (95-97%) but not erythroid Tfr2. As expected, circulating iron levels were increased in Tfr2-ASO mice, maintaining RBC count and Hb levels in the normal range for the first 2 weeks of treatment. However, Hb and RBCs reverted to control levels at 6 weeks, before the end of the protocol. These results show that increased iron availability alone, due to hepatic Tfr2 downregulation, delays anemia development but is not sufficient to boost erythropoiesis on a long term. In agreement, Tfr2KO mice, with Tfr2 inactivation both in the liver and in the erythroid compartment, maintained higher RBC count and Hb levels compared to controls until the end of the protocol. Conclusions The concomitant targeting of hepatic and erythroid Tfr2, here obtained through Tfr2 germline genetic inactivation, is necessary and sufficient to ameliorate anemia of CKD. On the contrary selective BM Tfr2 deletion that enhances EPO responsiveness of erythroid cells, or hepatic Tfr2 downregulation that increases iron availability, do not correct anemia in a long term. Therefore, a specific approach able to inhibit both hepatic and erythroid Tfr2 could adjust iron availability according to the enhanced erythropoiesis, correcting both drivers of anemia development in CKD. The development of a pharmacologic tool to downregulate Tfr2 might become an alternative to the standard treatment with ESAs plus iron supplementation with limited off-target effects due to the restricted TFR2 expression. Disclosures Aghajan: Ionis Pharmaceuticals, Inc.: Current Employment. Guo: Ionis Pharmaceuticals, Inc.: Current Employment.

β-thalassemia is an autosomal recessive disorder due to mutations in the β-globin gene, that leads to defective production of hemoglobin (Hb) and red blood cells (RBC). The main features of the disease are anemia, ineffective erythropoiesis and iron overload. Patients affected by the most severe form of β-thalassemia are transfusion-dependent (TDT) and require lifelong blood transfusions and iron chelation, symptomatic treatments that affect the quality of life. The only curative option, unfortunately limited by the insufficient number of HLA-matched donors, is allogenic bone marrow (BM) transplantation. Other recently approved treatments (i.e. Luspatercept and gene therapy) are only partially effective and/or suitable for selected patients. Thus, the identification of novel therapeutic approaches is a clinical need. Transferrin receptor 2 (TFR2) contributes to the transcriptional activation of hepcidin, the master regulator of iron homeostasis, in the liver and is a brake of Erythropoietin signaling in erythroid cells, thus balancing RBC production and iron availability. We have recently proved that BM Tfr2 deletion enhances erythropoiesis in wild-type mice (Nai et al., Blood 2015) and ameliorates anemia in non-TDT mice models, both alone (Artuso et al., Blood 2018) and in combination with iron-restricting agents (Casu, Pettinato et al., Blood 2020). Here we aim at investigating whether Tfr2 targeting might be beneficial also for TDT. To this purpose we generated TDT mice (Hbb th1/th2; Casu et al., Haematologica 2020) with heterozygous (Tfr2 BMhetero/Hbb th1/th2) and homozygous (Tfr2 BMKO/Hbb th1/th2) BM Tfr2 deletion by transplantation of Hbb th1/th2, Tfr2-hetero/Hbb th1/th2 and Tfr2-ko/Hbb th1/th2 fetal liver cells (FLT) from day E14.5 embryos into lethally irradiated wild-type mice. BM Tfr2 deficient mice have increased RBC count and Hb levels and decreased percentage of reticulocytes with a gene dosage effect 8 weeks after FLT, before the onset of transfusion-dependance. At this time-point, complete BM Tfr2 deletion ameliorates ineffective erythropoiesis, decreasing the percentage of polychromatic erythroblasts and increasing orthochromatic erythroblasts and mature RBCs both in the BM and in the spleen. The improved anemia was also accompanied by a partial correction of two debilitating TDT complications, iron overload and cardiomegaly. The beneficial effect of Tfr2 deletion was maintained over time. Indeed, Hbb th1/th2 mice became transfusion-dependent 14 weeks after FLT, when Hb levels drop below 5.5 g/dL, requiring transfusions of an average 108.75±56.87μL of blood/animal/week. On the contrary, animals with both heterozygous and homozygous BM Tfr2 deletion are still non-transfusion-dependent at 20 weeks, maintaining Hb levels above 7 and 9 g/dL respectively. Overall, our results prove that, despite the persistence of the genetic defect, hematopoietic Tfr2 deletion ameliorates anemia, ineffective erythropoiesis and secondary complications also in the most severe form of β-thalassemia. This improvement is associated to a substantial increase in transfusion-free survival of both Tfr2 BMhetero/Hbb th1/th2 and Tfr2 BMKO/Hbb th1/th2 mice, which are transfusion-independent 6 weeks after the time of blood transfusion requirement in Hbb th1/th2 animals. The difference of blood transfusions needs will be evaluated over time for at least 10 additional weeks. Our findings demonstrate that TFR2 targeting represents a new promising therapeutic opportunity for the management of β-thalassemia, worth to be tested both as a monotherapy and in association with available treatments. Disclosures Rivella: Incyte: Consultancy; MeiraGTx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Forma Theraputics: Consultancy; Keros Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ionis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Consultancy.

Gene therapy for hemoglobinopathies is currently based on transplantation of autologous hematopoietic stem cells genetically modified with a lentiviral vector expressing a globin gene under the control of globin transcriptional regulatory elements. Preclinical and early clinical studies showed the safety and potential efficacy of this therapeutic approach as well as the hurdles still limiting its general application. In addition, for both beta-thalassemia and sickle cell disease, an altered bone marrow microenvironment reduces the efficiency of stem cell harvesting as well as engraftment. These hurdles need be addressed for gene therapy for hemoglobinopathies to become a clinical reality.

International audience

In the last decade many studies unraveled the bone marrow (BM) niche regulation and crosstalk with hematopoietic stem cells (HSC) in steady state conditions and malignancies, but HSC-niche interactions are still underexplored in hematological inherited disorders. We have recently 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 congenital hemoglobin disorder resulting in severe anemia, ineffective erythropoiesis and multi-organ secondary complications, such as bone defects. It is one of the most globally widespread monogenic diseases, which can be cured by transplantation of HSC from compatible healthy donors or autologous HSC from patients upon gene therapy. Cases of graft failure have been reported, but causes have not been deeply investigated and might include an impaired HSC function and a defective supporting activity of the BM niche, worsened by age and disease progression. We showed that the prolonged residence of HSC into an altered BM stromal niche in BT Hbbth3/+ (th3) mice negatively affects stem cell number, quiescence and self-renewal. Moreover, we demonstrated that correction of HSC-stromal niche crosstalk rescues BT HSC function by in vivo reactivation of parathyroid hormone (PTH) signaling. Consistently with the common finding of osteoporosis in BT patients, we found reduced bone deposition and low levels of PTH also in the murine model. We investigated the potential mechanisms underlying the decreased PTH and bone defect and we focused on the role of fibroblast growth factor-23 (FGF-23). FGF-23 is a systemic hormone mainly secreted by osteocytes, which acts as negative regulator of bone metabolism by inhibiting bone mineralization and PTH production by parathyroid glands. Since FGF-23 is positively modulated by the anemia-related factor erythropoietin (EPO), we hypothesized that the high EPO levels in BT, subsequent to ineffective erythropoiesis, might contribute to increase FGF-23. We measured high levels of circulating FGF-23 in th3 mice (wt vs. th3: 399.7±69.77 vs. 1975±209.3 pg/ml, p Our findings uncover an underexplored role of FGF-23 in bone and BM niche defects in BT, as a condition of severe anemia and chronic EPO stimulation. The inhibition of FGF-23 signaling might provide a novel strategy to ameliorate bone compartment and restore HSC-BM niche interactions in BT, with a potential translational relevance in improving HSC transplantation approaches. Disclosures Motta: Sanofi Genzyme: Honoraria. Cappellini:BMS: Honoraria; CRISPR Therapeutics, Novartis, Vifor Pharma: Membership on an entity's Board of Directors or advisory committees; Genzyme/Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees.

β-thalassemias are genetic disorders characterized by anemia, ineffective erythropoiesis, and iron overload. Current treatment of severe cases is based on blood transfusion and iron chelation or allogeneic bone marrow (BM) transplantation. Novel approaches are explored for nontransfusion-dependent patients (thalassemia intermedia) who develop anemia and iron overload. Here, we investigated the erythropoietin (EPO) receptor partner, transferrin receptor 2 (TFR2), as a novel potential therapeutic target. We generated a murine model of thalassemia intermedia specifically lacking BM Tfr2: because their erythroid cells are more susceptible to EPO stimulation, mice show improved erythropoiesis and red blood cell morphology as well as partial correction of anemia and iron overload. The beneficial effects become attenuated over time, possibly due to insufficient iron availability to sustain the enhanced erythropoiesis. Germ line deletion of Tfr2, including haploinsufficiency, had a similar effect in the thalassemic model. Because targeting TFR2 enhances EPO-mediated effects exclusively in cells expressing both receptors, this approach may have advantages over erythropoiesis-stimulating agents in the treatment of other anemias.

Gene therapy for hemoglobinopathies is currently based on transplantation of autologous hematopoietic stem cells genetically modified with an integrating lentiviral vector expressing a globin gene under the control of globin transcriptional regulatory elements. Studies and safety works demonstrated the potential therapeutic efficacy and safety of this approach, providing the rationale for clinical translation. The outcomes of early clinical trials, although showing promising results, have highlighted the current limitations to a more general application. These include the nature, source and age of repopulating hematopoietic stem cells, the suboptimal transduction efficiency and gene expression levels, the toxicity and efficacy of bone marrow conditioning, the stress status of bone marrow microenvironment in chronic diseases such as β-thalassemia and sickle cell disease. Recently, gene editing strategies based on the use of nucleases offered a novel approach to increase globin expression in a quasi-physiological way, independently from the addition of transgenes and viral sequences to the human genome. This review will discuss the current status of gene therapy for β-thalassemia and sickle cell disease with a perspective towards the improvements necessary in the context of clinical translation.

Summary Ex vivo gene therapy based on CD34+ hematopoietic stem cells (HSCs) has shown promising results in clinical trials, but genetic engineering to high levels and in large scale remains challenging. We devised a sorting strategy that captures more than 90% of HSC activity in less than 10% of mobilized peripheral blood (mPB) CD34+ cells, and modeled a transplantation protocol based on highly purified, genetically engineered HSCs co-infused with uncultured progenitor cells. Prostaglandin E2 stimulation allowed near-complete transduction of HSCs with lentiviral vectors during a culture time of less than 38 hr, mitigating the negative impact of standard culture on progenitor cell function. Exploiting the pyrimidoindole derivative UM171, we show that transduced mPB CD34+CD38− cells with repopulating potential could be expanded ex vivo. Implementing these findings in clinical gene therapy protocols will improve the efficacy, safety, and sustainability of gene therapy and generate new opportunities in the field of gene editing., Highlights • CD34+CD38− cells as an HSC-enriched starting population for ex vivo gene therapy • Reduced culture time (, In this article, Gentner and colleagues undertake a comprehensive strategy to advance ex vivo genetic engineering of HSCs for gene therapy. They experimentally define an optimal strategy to purify HSCs, which allows uncoupling long-term from short-term hematopoietic reconstitution, and implement ex vivo conditions that best preserve their biological properties applying novel transduction-enhancing compounds and pyrimidoindole derivatives to support HSC expansion.

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.[1][1

Beta-thalassemia and sickle cell disease (SCD) are congenital anemias caused by mutations in the beta-globin gene, resulting in either reduced/absent production of globin chains or abnormal hemoglobin structure. At present, the definitive cure is represented by allogeneic hematopoietic stem cell transplantation, with a probability to find a well-matched donor of Experimental gene therapy for hemoglobinopathies is based on transplantation of autologous hematopoietic stem cells genetically modified to express therapeutic hemoglobin levels. Approaches to genetically modify HSCs for treatment of hemoglobinopathies include: 1) the addition of globin genes by lentiviral vectors and 2) gene editing by nucleases to reactivate fetal hemoglobin either through inhibition of repressors or by reproducing mutations associated with high fetal hemoglobin levels. The outcomes of early clinical trials are showing the safety and potential efficacy, as well as the hurdles still limiting a general application.Current challenges and improved strategies will be presented and discussed. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Plerixafor

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.

Genome-wide approaches allow investigating the molecular circuitry wiring the genetic and epigenetic programs of human somatic stem cells. Hematopoietic stem/progenitor cells (HSPC) give rise to the different blood cell types; however, the molecular basis of human hematopoietic lineage commitment is poorly characterized. Here, we define the transcriptional and epigenetic profile of human HSPC and early myeloid and erythroid progenitors by a combination of Cap Analysis of Gene Expression (CAGE), ChIP-seq and Moloney leukemia virus (MLV) integration site mapping. Most promoters and transcripts were shared by HSPC and committed progenitors, while enhancers and super-enhancers consistently changed upon differentiation, indicating that lineage commitment is essentially regulated by enhancer elements. A significant fraction of CAGE promoters differentially expressed upon commitment were novel, harbored a chromatin enhancer signature, and may identify promoters and transcribed enhancers driving cell commitment. MLV-targeted genomic regions co-mapped with cell-specific active enhancers and super-enhancers. Expression analyses, together with an enhancer functional assay, indicate that MLV integration can be used to identify bona fide developmentally regulated enhancers. Overall, this study provides an overview of transcriptional and epigenetic changes associated to HSPC lineage commitment, and a novel signature for regulatory elements involved in cell identity.

Detection of hemoglobin (Hb) variants represents an important issue for diagnosis and adequate treatment of hemoglobinopathies. The Capillarys 2 Flex Piercing analyzer (Capillarys) by Sebia is routinely used in our clinical laboratories to detect Hb variants in peripheral blood (PB). This automated method separates Hb fractions by capillary electrophoresis, giving a spectrophotometric measure of their relative proportion. The scientific research in the field of hemoglobinopathies needs robust procedures to evaluate the efficacy of experimental therapies, as gene therapy. We investigated for the first time the feasibility to use Capillarys on cellular lysates from in vitro erythroid cultures. Because total Hb concentration in erythroid lysates is up to 20-fold lower than in hemolysates from PB, we analyzed diluted blood samples, thanks to the manual mode included in the Capillarys setting. We compared analytical precision, accuracy, sensitivity, and specificity of this procedure to the automatic method, routinely used in diagnostics. For instance, adult Hb intra- and interassay precision were estimated as coefficient of variation 0.2% and 0.3%, respectively. The manual mode is less robust for detection of fractions

Sox6 belongs to the Sry (sex-determining region Y)–related high-mobility-group–box family of transcription factors, which control cell-fate specification of many cell types. Here, we explored the role of Sox6 in human erythropoiesis by its overexpression both in the erythroleukemic K562 cell line and in primary erythroid cultures from human cord blood CD34+ cells. Sox6 induced significant erythroid differentiation in both models. K562 cells underwent hemoglobinization and, despite their leukemic origin, died within 9 days after transduction; primary erythroid cultures accelerated their kinetics of erythroid maturation and increased the number of cells that reached the final enucleation step. Searching for direct Sox6 targets, we found SOCS3 (suppressor of cytokine signaling-3), a known mediator of cytokine response. Sox6 was bound in vitro and in vivo to an evolutionarily conserved regulatory SOCS3 element, which induced transcriptional activation. SOCS3 overexpression in K562 cells and in primary erythroid cells recapitulated the growth inhibition induced by Sox6, which demonstrates that SOCS3 is a relevant Sox6 effector.

Transferrin receptor 2 (TFR2), the type 3 hemochromatosis gene, is an activator of the iron hormone hepcidin in the liver and a partner of erythropoietin (EPO) receptor in erythroid cells. The loss of bone marrow (BM) Tfr2 increases erythroblast EPO sensitivity inducing erythrocytosis in mice (Nai et al, Blood 2015). We explored whether deletion of BM Tfr2 improves anemia and ineffective erythropoiesis in β-thalassemias, iron-loading anemias due to recessive β-globin gene mutations. We generated thalassemic mice (Hbbth3/+) with selective BM inactivation of Tfr2 (Tfr2BMKO/Hbbth3/+) through BM transplantation (BMT). Deletion of BM Tfr2 ameliorates RBC morphology with consistent and persistent increase of RBC count and Hb levels in thalassemic mice, accompanied by reduced iron accumulation. Around 22 weeks after BMT the improvement fades in double mutantanimals: Hb levels return comparable to those of Hbbth3/+mice, while RBC count persists higher. Anemia improvement in double mutant mice reduces serum EPO levels and improves erythropoiesis, in particular 22 weeks after BMT. Overall these data prove that the loss of the beneficial effect of deleting Tfr2 is not accounted for by erythropoiesis failure, but likely by exhaustion of splenic iron consumed by the enhanced erythropoiesis. In order to elucidate the molecular mechanisms of the phenotype improvement, we investigated whether the EPO-EPOR signaling pathway is overactive, as occurs in Tfr2 null erythroid cells (Nai et al, Blood 2015). Taking into account that Tfr2BMKO/Hbbth3/+ mice have lower serum EPO than Hbbth3/+, the expression levels of target genes of the EPOR-JAK2-STAT5 (Erfe and Bcl-xl) and of EPOR-PI3K-AKT pathway (Fasl, Epor and Ccng2) is consistent with the signaling being inappropriately active in double mutant mice. To start unraveling the global molecular/cellular processes underlying the remarkable phenotype amelioration, we performed RNAseq analysis on spleen samples from double mutant and Hbbth3/+ control mice at the time point of maximal erythropoiesis improvement (22 weeks post BMT). Spleens are enlarged in both genotypes with about 80% Ter119+ (erythroid) cells in both. In total we identified 2796 genes (1997 protein coding) differentially regulated between the two genotypes. The analysis of iron-related genes reveals a strong reduction of the expression of the iron exporter Fpn, Hmox1 and Alas2, suggestive of decreased hemolysis and/or of free heme accumulation in double mutants. Gene ontology analysis reveals enrichment of genes involved in cell cycle and proliferation, mitochondrial function, as well as proteasome activity and of most of the antioxidant targets (Sod1, Sod2, Fth1, Txn1, Txn2, Gstpi) of the canonical NF-kB pathway. Underrepresented genes are those involved in lipid handling, leukocyte/lymphocyte differentiation and coagulation. In summary, the RNAseq patterns indicate an increased spleen erythroid commitment and a mitochondrial metabolic shift, similar to the shift occurring during hematopoietic development to sustain erythroid proliferation and differentiation. We speculate that this effect is mediated by the enhanced EPO sensitivity. Interestingly EPO directly stimulates mitochondrial genes expression in adipocytes. Also the increased proteasome activity may significantly contribute to the improved erythropoiesis, since proteasomal degradation is required in the process of erythroblast enucleation. Finally, the activation of the NF-kB antioxidant response may contrast ROS increase and limit ineffective erythropoiesis. In conclusion, targeting erythroid TFR2 might become a novel erythropoiesis stimulating approach, worth to be tested in other forms of anemia. Disclosures Muckenthaler: Novartis: Research Funding. Camaschella:vifor Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Bone marrow (BM) contains a population of mesenchymal stromal cells (MSC) that, together with endothelial cells and osteoblasts, provide a specific microenvironment to support hematopoietic stem cell homeostasis. Beta-thalassemia (BT) is an hereditary blood disorder characterized by reduced or absent synthesis of hemoglobin beta-chains. Data on the mesenchymal compartment in BT patients are scarce. We isolated and characterized MSCs from BT and healthy donor (HD) BM samples. BT-MSCs showed a reduced clonogenic capacity, delay in colony formation, lower numbers of CFU-Fs and longer population doubling time. Similarly, we observed an altered differentiation capacity into adipocytes and osteoblasts. Both HD- and BT-MSCs express the canonical mesenchymal markers. On the contrary, the expression of CD146 and CD271 was extremely reduced in BT-MSCs, indicating a pauperization of the most primitive stem cell pool. We analyzed the expression of genes involved in the crosstalk between MSCs and HSCs and found a reduced expression of Cxcl12, SCF and Angp1 in BT-MSCs. Several genes relevant for MSC functionality were also downregulated in BT-MSCs. We demonstrated that MSCs are able to uptake and store iron. ROS levels were increased in BT-MSCs possibly due to altered anti-oxidant response and iron overload associated with blood transfusions. Importantly, higher level of ROS and reduced expression of primitive markers were observed in HD-MSCs cultured in presence of iron, underlying the importance of iron level for normal MSC function. In conclusion, we showed an impairment in the mesenchymal niche of BT-BM possibly associated with prolonged iron exposure. Disclosures Marktel: GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding. Ciceri: GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding.

Lentiviral (LV)-based hematopoietic stem and progenitor cell (HSPC) gene therapy is becoming a promising alternative to allogeneic stem cell transplantation for curing genetic diseases. To potentially improve the efficacy, safety and economic sustainability of HSPC transduction, we reasoned to genetically manipulate only the more potent CD34+CD38- HSPC, thereby improving HSPC maintenance in culture in the absence of differentiating cells and downscaling the cell therapy product by a factor of ten without compromising long-term engraftment. This approach would also decrease the total load of vector integration infused in the patients, thus improving its overall safety.First, we determined the engraftment kinetics of CD34+ mobilized peripheral blood (mPB) subpopulations over a 24wk xenotransplantation period. We sorted CD34+ mPB into 4 fractions with increasing expression levels of CD38 and marked each fraction with a specific fluorescent protein allowing to track the population of origin driving hematopoietic reconstitution. Differentially labeled fractions were mixed, and various combinations of CD38-, CD38int and CD38hi HSPC were injected into NSG mice (2 exp, n=30). Almost all long-term repopulating capacity (>90%) was contained within CD34+CD38- cells, and these cells took over hematopoiesis by 9wks. Instead, early reconstitution was mainly driven by CD34+CD38int progenitor cells.In the prospect of a clinical translation, we then modeled the co-administration of gene-modified CD34+CD38- mPB cells with uncultured CD34+CD38int/+ supporter cells aimed to drive fast hematopoietic recovery (3 exp, n=38). Repopulation by gene-modified CD34+CD38- cells was slower (15wks) and incomplete ( 5 wks and re-established long-term (24wks) gene marking up to 85%, thus allowing to benefit from prompt hematopoietic recovery driven by transiently repopulating CD38int/+ supporter cells.Last, we optimized LV transduction in the framework of an improved culture protocol. Exposing CD34+ or CD34+CD38- mPB cells to prostaglandin E2 (PGE2) increased transduction efficiency 1.5-2.5x, allowing to markedly reduce pre-stimulation and LV exposure times better preserving HSC functions. Importantly, the higher gene-transfer efficiency was maintained for up to 24 wks following xenotransplantation (n=33), suggesting that PGE2 facilitated LV transduction in long-term HSC.In summary, these results support the clinical development of novel HSPC gene therapy protocols based on the modification of highly purified HSC subsets, with the prospect to improve the efficacy, safety and feasibility of future ex vivo gene therapy studies.