Droplet digital polymerase chain reaction-based quantitation of therapeutic lentiviral vector copies in transduced hematopoietic stem cells.

BACKGROUND AIMS: Gene therapy using lentiviral vectors (LVs) that harbor a functional ß-globin gene provides a curative treatment for hemoglobinopathies including beta-thalassemia and sickle cell disease. Accurate quantification of the vector copy number (VCN) and/or the proportion of transduced cells is critical to evaluate the efficacy of transduction and stability of the transgene during treatment. Moreover, commonly used techniques for LV quantification, including real-time quantitative polymerase chain reaction (PCR) or fluorescence-activated cell sorting, require either a standard curve or expression of a reporter protein for the detection of transduced cells. In the present study, we describe a digital droplet PCR (ddPCR) technique to measure the lentiviral VCN in transduced hematopoietic stem and progenitor cells (HSPCs). METHODS: After HSPCs were transduced with an LV encoding the therapeutic ß-globin (ßA-T87Q) gene, the integrated lentiviral sequence in the host genome was amplified with primers that targeted a sequence within the vector and the human RPP30 gene. The dynamic range of ddPCR was between 5 × 10-3 ng and 5 × 10-6 ng of target copy per reaction. RESULTS: We found that the ddPCR-based approach was able to estimate VCN with high sensitivity and a low standard deviation. Furthermore, ddPCR-mediated quantitation of lentiviral copy numbers in differentiated erythroblasts correlated with the level of ßA-T87Q protein detected by reverse-phase high-performance liquid chromatography. CONCLUSIONS: Taken together, the ddPCR technique has the potential to precisely detect LV copy numbers in the host genome, which can be used for VCN estimation, calculation of infectious titer and multiplicity of infection for HSPC transduction in a clinical setting.

Lovo-cel gene therapy for sickle cell disease: Treatment process evolution and outcomes in the initial groups of the HGB-206 study.

lovo-cel (bb1111; LentiGlobin for sickle cell disease [SCD]) gene therapy (GT) comprises autologous transplantation of hematopoietic stem and progenitor cells transduced with the BB305 lentiviral vector encoding a modified ß-globin gene (ßA-T87Q ) to produce anti-sickling hemoglobin (HbAT87Q ). The efficacy and safety of lovo-cel for SCD are being evaluated in the ongoing phase 1/2 HGB-206 study (ClinicalTrials.gov: NCT02140554). The treatment process evolved over time, using learnings from outcomes in the initial patients to optimize lovo-cel's benefit-risk profile. Following modest expression of HbAT87Q in the initial patients (Group A, n = 7), alterations were made to the treatment process for patients subsequently enrolled in Group B (n = 2, patients B1 and B2), including improvements to cell collection and lovo-cel manufacturing. After 6 months, median Group A peripheral blood vector copy number (≥0.08 c/dg) and HbAT87Q levels (≥0.46 g/dL) were inadequate for substantial clinical effect but stable and sustained over 5.5 years; both markedly improved in Group B (patient B1: ≥0.53 c/dg and ≥2.69 g/dL; patient B2: ≥2.14 c/dg and ≥6.40 g/dL, respectively) and generated improved biologic and clinical efficacy in Group B, including higher total hemoglobin and decreased hemolysis. The safety of the lovo-cel for SCD treatment regimen largely reflected the known side effects of HSPC collection, busulfan conditioning regimen, and underlying SCD; acute myeloid leukemia was observed in two patients in Group A and deemed unlikely related to insertional oncogenesis. Changes made during development of the lovo-cel treatment process were associated with improved outcomes and provide lessons for future SCD GT studies.

Erythropoietin and IGF-1 signaling synchronize cell proliferation and maturation during erythropoiesis.

Tight coordination of cell proliferation and differentiation is central to red blood cell formation. Erythropoietin controls the proliferation and survival of red blood cell precursors, while variations in GATA-1/FOG-1 complex composition and concentrations drive their maturation. However, clear evidence of cross-talk between molecular pathways is lacking. Here, we show that erythropoietin activates AKT, which phosphorylates GATA-1 at Ser310, thereby increasing GATA-1 affinity for FOG-1. In turn, FOG-1 displaces pRb/E2F-2 from GATA-1, ultimately releasing free, proproliferative E2F-2. Mice bearing a Gata-1(S310A) mutation suffer from fatal anemia when a compensatory pathway for E2F-2 production involving insulin-like growth factor-1 (IGF-1) signaling is simultaneously abolished. In the context of the GATA-1(V205G) mutation resulting in lethal anemia, we show that the Ser310 cannot be phosphorylated and that constitutive phosphorylation at this position restores partial erythroid differentiation. This study sheds light on the GATA-1 pathways that synchronize cell proliferation and differentiation for tissue homeostasis.

Coordinated ß-globin expression and α2-globin reduction in a multiplex lentiviral gene therapy vector for ß-thalassemia.

A primary challenge in lentiviral gene therapy of ß-hemoglobinopathies is to maintain low vector copy numbers to avoid genotoxicity while being reliably therapeutic for all genotypes. We designed a high-titer lentiviral vector, LVß-shα2, that allows coordinated expression of the therapeutic ßA-T87Q-globin gene and of an intron-embedded miR-30-based short hairpin RNA (shRNA) selectively targeting the α2-globin mRNA. Our approach was guided by the knowledge that moderate reduction of α-globin chain synthesis ameliorates disease severity in ß-thalassemia. We demonstrate that LVß-shα2 reduces α2-globin mRNA expression in erythroid cells while keeping α1-globin mRNA levels unchanged and ßA-T87Q-globin gene expression identical to the parent vector. Compared with the first ßA-T87Q-globin lentiviral vector that has received conditional marketing authorization, BB305, LVß-shα2 shows 1.7-fold greater potency to improve α/ß ratios. It may thus result in greater therapeutic efficacy and reliability for the most severe types of ß-thalassemia and provide an improved benefit/risk ratio regardless of the ß-thalassemia genotype.

Successful Derivation of Hepatoblasts, Cholangiocytes and Hepatocytes from Simian Induced Pluripotent Stem Cells.

The use of primary cells in human liver therapy is limited by a lack of cells. Induced pluripotent stem cells (iPSCs) represent an alternative to primary cells as they are infinitely expandable and can be differentiated into different liver cell types. The aim of our work was to demonstrate that simian iPSCs (siPSCs) could be used as a new source of liver cells to be used as a large animal model for preclinical studies. We first differentiated siPSCs into a homogenous population of hepatoblasts (siHBs). We then separately differentiated them into hepatocytes (siHeps) and cholangiocytes (siChols) expressing respective specific markers and displaying epithelial polarity. Moreover, we showed that polarized siChols can self-organize into 3D structures. These results should facilitate the deciphering of liver development and open the way to exploring co-culture systems that could be assessed during preclinical studies, including in autologous monkey donors, for regenerative medicine purposes.

Gene Therapy in Patients with Transfusion-Dependent ß-Thalassemia.

BACKGROUND: Donor availability and transplantation-related risks limit the broad use of allogeneic hematopoietic-cell transplantation in patients with transfusion-dependent ß-thalassemia. After previously establishing that lentiviral transfer of a marked ß-globin (ßA-T87Q) gene could substitute for long-term red-cell transfusions in a patient with ß-thalassemia, we wanted to evaluate the safety and efficacy of such gene therapy in patients with transfusion-dependent ß-thalassemia. METHODS: In two phase 1-2 studies, we obtained mobilized autologous CD34+ cells from 22 patients (12 to 35 years of age) with transfusion-dependent ß-thalassemia and transduced the cells ex vivo with LentiGlobin BB305 vector, which encodes adult hemoglobin (HbA) with a T87Q amino acid substitution (HbAT87Q). The cells were then reinfused after the patients had undergone myeloablative busulfan conditioning. We subsequently monitored adverse events, vector integration, and levels of replication-competent lentivirus. Efficacy assessments included levels of total hemoglobin and HbAT87Q, transfusion requirements, and average vector copy number. RESULTS: At a median of 26 months (range, 15 to 42) after infusion of the gene-modified cells, all but 1 of the 13 patients who had a non-ß0/ß0 genotype had stopped receiving red-cell transfusions; the levels of HbAT87Q ranged from 3.4 to 10.0 g per deciliter, and the levels of total hemoglobin ranged from 8.2 to 13.7 g per deciliter. Correction of biologic markers of dyserythropoiesis was achieved in evaluated patients with hemoglobin levels near normal ranges. In 9 patients with a ß0/ß0 genotype or two copies of the IVS1-110 mutation, the median annualized transfusion volume was decreased by 73%, and red-cell transfusions were discontinued in 3 patients. Treatment-related adverse events were typical of those associated with autologous stem-cell transplantation. No clonal dominance related to vector integration was observed. CONCLUSIONS: Gene therapy with autologous CD34+ cells transduced with the BB305 vector reduced or eliminated the need for long-term red-cell transfusions in 22 patients with severe ß-thalassemia without serious adverse events related to the drug product. (Funded by Bluebird Bio and others; HGB-204 and HGB-205 ClinicalTrials.gov numbers, NCT01745120 and NCT02151526 .).

Erosion of the chronic myeloid leukaemia stem cell pool by PPARγ agonists.

Whether cancer is maintained by a small number of stem cells or is composed of proliferating cells with approximate phenotypic equivalency is a central question in cancer biology. In the stem cell hypothesis, relapse after treatment may occur by failure to eradicate cancer stem cells. Chronic myeloid leukaemia (CML) is quintessential to this hypothesis. CML is a myeloproliferative disorder that results from dysregulated tyrosine kinase activity of the fusion oncoprotein BCR-ABL. During the chronic phase, this sole genetic abnormality (chromosomal translocation Ph(+): t(9;22)(q34;q11)) at the stem cell level causes increased proliferation of myeloid cells without loss of their capacity to differentiate. Without treatment, most patients progress to the blast phase when additional oncogenic mutations result in a fatal acute leukaemia made of proliferating immature cells. Imatinib mesylate and other tyrosine kinase inhibitors (TKIs) that target the kinase activity of BCR-ABL have improved patient survival markedly. However, fewer than 10% of patients reach the stage of complete molecular response (CMR), defined as the point when BCR-ABL transcripts become undetectable in blood cells. Failure to reach CMR results from the inability of TKIs to eradicate quiescent CML leukaemia stem cells (LSCs). Here we show that the residual CML LSC pool can be gradually purged by the glitazones, antidiabetic drugs that are agonists of peroxisome proliferator-activated receptor-γ (PPARγ). We found that activation of PPARγ by the glitazones decreases expression of STAT5 and its downstream targets HIF2α and CITED2, which are key guardians of the quiescence and stemness of CML LSCs. When pioglitazone was given temporarily to three CML patients in chronic residual disease in spite of continuous treatment with imatinib, all of them achieved sustained CMR, up to 4.7 years after withdrawal of pioglitazone. This suggests that clinically relevant cancer eradication may become a generally attainable goal by combination therapy that erodes the cancer stem cell pool.

Gene Therapy in a Patient with Sickle Cell Disease.

Sickle cell disease results from a homozygous missense mutation in the ß-globin gene that causes polymerization of hemoglobin S. Gene therapy for patients with this disorder is complicated by the complex cellular abnormalities and challenges in achieving effective, persistent inhibition of polymerization of hemoglobin S. We describe our first patient treated with lentiviral vector-mediated addition of an antisickling ß-globin gene into autologous hematopoietic stem cells. Adverse events were consistent with busulfan conditioning. Fifteen months after treatment, the level of therapeutic antisickling ß-globin remained high (approximately 50% of ß-like-globin chains) without recurrence of sickle crises and with correction of the biologic hallmarks of the disease. (Funded by Bluebird Bio and others; HGB-205 ClinicalTrials.gov number, NCT02151526 .).

HSP70 sequestration by free α-globin promotes ineffective erythropoiesis in ß-thalassaemia.

ß-Thalassaemia major (ß-TM) is an inherited haemoglobinopathy caused by a quantitative defect in the synthesis of ß-globin chains of haemoglobin, leading to the accumulation of free α-globin chains that form toxic aggregates. Despite extensive knowledge of the molecular defects causing ß-TM, little is known of the mechanisms responsible for the ineffective erythropoiesis observed in the condition, which is characterized by accelerated erythroid differentiation, maturation arrest and apoptosis at the polychromatophilic stage. We have previously demonstrated that normal human erythroid maturation requires a transient activation of caspase-3 at the later stages of maturation. Although erythroid transcription factor GATA-1, the master transcriptional factor of erythropoiesis, is a caspase-3 target, it is not cleaved during erythroid differentiation. We have shown that, in human erythroblasts, the chaperone heat shock protein70 (HSP70) is constitutively expressed and, at later stages of maturation, translocates into the nucleus and protects GATA-1 from caspase-3 cleavage. The primary role of this ubiquitous chaperone is to participate in the refolding of proteins denatured by cytoplasmic stress, thus preventing their aggregation. Here we show in vitro that during the maturation of human ß-TM erythroblasts, HSP70 interacts directly with free α-globin chains. As a consequence, HSP70 is sequestrated in the cytoplasm and GATA-1 is no longer protected, resulting in end-stage maturation arrest and apoptosis. Transduction of a nuclear-targeted HSP70 mutant or a caspase-3-uncleavable GATA-1 mutant restores terminal maturation of ß-TM erythroblasts, which may provide a rationale for new targeted therapies of ß-TM.

The integrity of the FOG-2 LXCXE pRb-binding motif is required for small intestine homeostasis.

NEW FINDINGS: What is the central question of this study? Do Fog2Rb-/Rb- mice present a defect of small intestine homeostasis? What is the main finding and its importance? The importance of interactions between FOG-2 and pRb in adipose tissue physiology has previously been demonstrated. Here it is shown that this interaction is also intrinsic to small intestine homeostasis and exerts extrinsic control over mouse metabolism. Thus, this association is involved in maintaining small intestine morphology, and regulating crypt proliferation and lineage differentiation. It therefore affects mouse growth and adaptation to a high-fat diet. ABSTRACT: GATA transcription factors and their FOG cofactors play a key role in tissue-specific development and differentiation, from worms to humans. We have shown that GATA-1 and FOG-2 contain an LXCXE pRb-binding motif. Interactions between retinoblastoma protein (pRb) and GATA-1 are crucial for erythroid proliferation and differentiation, whereas the LXCXE pRb-binding site of FOG-2 is involved in adipogenesis. Fog2-knock-in mice have defective pRb binding and are resistant to obesity, due to efficient white-into-brown fat conversion. Our aim was to investigate the pathophysiological impact of FOG-2-pRb interaction on the small intestine and mouse growth. Histological analysis of the small intestine revealed architectural changes in Fog2Rb-/Rb- mice, including villus shortening, with crypt expansion and a change in muscularis propria thickness. These differences were more marked in the proximo-distal part of the small intestine and were associated with an increase in crypt cell proliferation and disruption of the goblet and Paneth cell lineage. The small intestine of the mutants was unable to adapt to a high-fat diet, and had significantly lower plasma lipid levels on such a diet. Fog2Rb-/Rb- mice displayed higher levels of glucose-dependent insulinotropic peptide release, and lower levels of insulin-like growth factor I release on a regular diet. Their intestinal lipid absorption was impaired, resulting in restricted weight gain. In addition to the intrinsic effects of the mutation on adipose tissue, we show here an extrinsic relationship between the intestine and the effect of FOG-2 mutation on mouse metabolism. In conclusion, the interaction of FOG-2 with pRb coordinates the crypt-villus axis and controls small intestine homeostasis.

Pioglitazone together with imatinib in chronic myeloid leukemia: A proof of concept study.

BACKGROUND: We recently reported that peroxisome proliferator-activated receptor γ agonists target chronic myeloid leukemia (CML) quiescent stem cells in vitro by decreasing transcription of STAT5. Here in the ACTIM phase 2 clinical trial, we asked whether pioglitazone add-on therapy to imatinib would impact CML residual disease, as assessed by BCR-ABL1 transcript quantification. METHODS: CML patients were eligible if treated with imatinib for at least 2 years at a stable daily dose, having yielded major molecular response (MMR) but not having achieved molecular response 4.5 (MR4.5 ) defined by BCR-ABL1/ABL1IS RNA levels ≤ 0.0032%. After inclusion, patients started pioglitazone at a dosage of 30 to 45 mg/day in addition to imatinib. The primary objective was to evaluate the cumulative incidence of patients having progressed from MMR to MR4.5 over 12 months. RESULTS: Twenty-four patients were included (age range, 24-79 years). No pharmacological interaction was observed between the drugs. The main adverse events were weight gain in 12 patients and a mean decrease of 0.4 g/dL in hemoglobin concentration. The cumulative incidence of MR4.5 was 56% (95% confidence interval, 37%-76%) by 12 months, despite a wide range of therapy duration (1.9-15.5 months), and 88% of 17 evaluable patients who were still on imatinib reached MR4.5 by 48 months. The cumulative incidence of MMR to MR4.5 spontaneous conversions over 12 months was estimated to be 23% with imatinib alone in a parallel cohort of patients. CONCLUSION: Pioglitazone in combination with imatinib was well tolerated and yielded a favorable 56% rate. These results provide a proof of concept needing confirmation within a randomized clinical trial (EudraCT 2009-011675-79). Cancer 2017;123:1791-1799. © 2016 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Inhibition of the acetyl lysine-binding pocket of bromodomain and extraterminal domain proteins interferes with adipogenesis.

The bromodomain and extraterminal (BET) domain family proteins are epigenetic modulators involved in the reading of acetylated lysine residues. The first BET protein inhibitor to be identified, (+)-JQ1, a thienotriazolo-1, 4-diazapine, binds selectively to the acetyl lysine-binding pocket of BET proteins. We evaluated the impact on adipogenesis of this druggable targeting of chromatin epigenetic readers, by investigating the physiological consequences of epigenetic modifications through targeting proteins binding to chromatin. JQ1 significantly inhibited the differentiation of 3T3-L1 preadipocytes into white and brown adipocytes by down-regulating the expression of genes involved in adipogenesis, particularly those encoding the peroxisome proliferator-activated receptor (PPAR-γ), the CCAAT/enhancer-binding protein (C/EBPα) and, STAT5A and B. The expression of a constitutively activated STAT5B mutant did not prevent inhibition by JQ1. Thus, the association of BET/STAT5 is required for adipogenesis but STAT5 transcription activity is not the only target of JQ1. Treatment with JQ1 did not lead to the conversion of white adipose tissue into brown adipose tissue (BAT). BET protein inhibition thus interferes with generation of adipose tissue from progenitors, confirming the importance of the connections between epigenetic mechanisms and specific adipogenic transcription factors.

Transfusion independence and HMGA2 activation after gene therapy of human ß-thalassaemia.

The ß-haemoglobinopathies are the most prevalent inherited disorders worldwide. Gene therapy of ß-thalassaemia is particularly challenging given the requirement for massive haemoglobin production in a lineage-specific manner and the lack of selective advantage for corrected haematopoietic stem cells. Compound ß(E)/ß(0)-thalassaemia is the most common form of severe thalassaemia in southeast Asian countries and their diasporas. The ß(E)-globin allele bears a point mutation that causes alternative splicing. The abnormally spliced form is non-coding, whereas the correctly spliced messenger RNA expresses a mutated ß(E)-globin with partial instability. When this is compounded with a non-functional ß(0) allele, a profound decrease in ß-globin synthesis results, and approximately half of ß(E)/ß(0)-thalassaemia patients are transfusion-dependent. The only available curative therapy is allogeneic haematopoietic stem cell transplantation, although most patients do not have a human-leukocyte-antigen-matched, geno-identical donor, and those who do still risk rejection or graft-versus-host disease. Here we show that, 33 months after lentiviral ß-globin gene transfer, an adult patient with severe ß(E)/ß(0)-thalassaemia dependent on monthly transfusions since early childhood has become transfusion independent for the past 21 months. Blood haemoglobin is maintained between 9 and 10 g dl(-1), of which one-third contains vector-encoded ß-globin. Most of the therapeutic benefit results from a dominant, myeloid-biased cell clone, in which the integrated vector causes transcriptional activation of HMGA2 in erythroid cells with further increased expression of a truncated HMGA2 mRNA insensitive to degradation by let-7 microRNAs. The clonal dominance that accompanies therapeutic efficacy may be coincidental and stochastic or result from a hitherto benign cell expansion caused by dysregulation of the HMGA2 gene in stem/progenitor cells.

Deficiency of the cytoskeletal protein SPECC1L leads to oblique facial clefting.

Genetic mutations responsible for oblique facial clefts (ObFC), a unique class of facial malformations, are largely unknown. We show that loss-of-function mutations in SPECC1L are pathogenic for this human developmental disorder and that SPECC1L is a critical organizer of vertebrate facial morphogenesis. During murine embryogenesis, Specc1l is expressed in cell populations of the developing facial primordial, which proliferate and fuse to form the face. In zebrafish, knockdown of a SPECC1L homolog produces a faceless phenotype with loss of jaw and facial structures, and knockdown in Drosophila phenocopies mutants in the integrin signaling pathway that exhibit cell-migration and -adhesion defects. Furthermore, in mammalian cells, SPECC1L colocalizes with both tubulin and actin, and its deficiency results in defective actin-cytoskeleton reorganization, as well as abnormal cell adhesion and migration. Collectively, these data demonstrate that SPECC1L functions in actin-cytoskeleton reorganization and is required for proper facial morphogenesis.

Arrayed lentiviral barcoding for quantification analysis of hematopoietic dynamics.

Our understanding of system dynamics of mixed cell populations in whole organisms has benefited from the advent of individual cell marking by nonarrayed DNA barcodes subsequently analyzed by high-throughput DNA sequencing. However, key limitations include statistical biases compromising quantification and the lack of applicability to deconvolute individual cell fate in vivo after pooling single cells differentially exposed to different conditions ex vivo. Here, we have derived an arrayed lentiviral library of DNA barcodes and obtained a proof-of-concept of its resolving capacity by quantifying hematopoietic regeneration after engraftment of mice with genetically modified autologous cells. This method has helped clarify and bridge the seemingly opposed clonal-succession and continuous-recruitment models of hematopoietic stem cell behavior and revealed that myeloid-lymphoid biases are common occurrences in steady-state hematopoiesis. Arrayed lentiviral barcoding should prove a versatile and powerful approach to deconvolute cell dynamics in vivo with applications in hematology, embryology, and cancer biology.

Parallel assessment of globin lentiviral transfer in induced pluripotent stem cells and adult hematopoietic stem cells derived from the same transplanted ß-thalassemia patient.

A patient with ß(E)/ß(0) -thalassemia major was converted to transfusion-independence 4.5 years ago by lentiviral gene transfer in hematopoietic stem cells while showing a myeloid-biased cell clone. Induced pluripotent stem cells (iPSCs) are a potential alternative source of hematopoietic stem cells. If fetal to adult globin class, switching does not occur in vivo in iPSC-derived erythroid cells, ß-globin gene transfer would be unnecessary. To investigate both vector integration skewing and the potential use of iPSCs for the treatment of thalassemia, we derived iPSCs from the thalassemia gene therapy patient and compared iPSC-derived hematopoietic cells to their natural isogenic somatic counterparts. In NSG immunodeficient mice, embryonic to fetal and a partial fetal to adult globin class switching were observed, indicating that the gene transfer is likely necessary for iPSC-based therapy of the ß-hemoglobinopathies. Lentivector integration occurred in regions of low and high genotoxicity. Surprisingly, common integration sites (CIS) were identified across those iPSCs and cells retrieved from isogenic and nonisogenic gene therapy patients with ß-thalassemia and adrenoleukodystrophy, respectively. This suggests that CIS observed in the absence of overt tumorigenesis result from nonrandom lentiviral integration rather than oncogenic in vivo selection. These findings bring the use of iPSCs closer to practicality and further clarify our interpretation of genome-wide lentivector integration.

Is normal hematopoiesis maintained solely by long-term multipotent stem cells?

The understanding of the hierarchical organization of the human hematopoietic system is of major biologic and clinical significance. The validity of the conventional model in which hematopoiesis is solely maintained by a pool of multipotent long-term hematopoietic stem cells (LT-HSCs) has been recently challenged by several mouse studies. These new data point to the existence of a heterogeneous stem cell population that consists of distinct subsets of LT-HSCs, which include stem cells biased toward lineage-specific differentiation programs. This review attempts to discuss the balanced versus biased patterns of lineage output of human LT-HSCs gathered in 3 different gene therapy trials on the basis of vector integration site analysis by deep sequencing. The distribution of integration sites observed tends to support the validity of the revised model.

Correction of murine ß-thalassemia after minimal lentiviral gene transfer and homeostatic in vivo erythroid expansion.

A challenge for gene therapy of genetic diseases is to maintain corrected cell populations in subjects undergoing transplantation in cases in which the corrected cells do not have intrinsic selective advantage over nontransduced cells. For inherited hematopoietic disorders, limitations include inefficient transduction of stem cell pools, the requirement for toxic myelosuppression, and a lack of optimal methods for cell selection after transduction. Here, we have designed a lentiviral vector that encodes human ß-globin and a truncated erythropoietin receptor, both under erythroid-specific transcriptional control. This truncated receptor confers enhanced sensitivity to erythropoietin and a benign course in human carriers. Transplantation of marrow transduced with the vector into syngenic thalassemic mice, which have elevated plasma erythropoietin levels, resulted in long-term correction of the disease even at low ratios of transduced/untransduced cells. Amplification of the red over the white blood cell lineages was self-controlled and averaged ∼ 100-fold instead of ∼ 5-fold for ß-globin expression alone. There was no detectable amplification of white blood cells or alteration of hematopoietic homeostasis. Notwithstanding legitimate safety concerns in the context of randomly integrating vectors, this approach may prove especially valuable in combination with targeted integration or in situ homologous recombination/repair and may lower the required level of pretransplantation myelosuppression.