[Show abstract][Hide abstract] ABSTRACT: RNA delivery is an attractive strategy to achieve transient gene expression in research projects and in cell- or gene-based therapies. Despite significant efforts investigating vector-directed RNA transfer, there is still a requirement for better efficiency of delivery to primary cells and in vivo. Retroviral platforms drive RNA delivery, yet retrovirus RNA-packaging constraints limit gene transfer to two genome-molecules per viral particle. To improve retroviral transfer, we designed a dimerization-independent MS2-driven RNA packaging system using MS2-Coat-retrovirus chimeras. The engineered chimeric particles promoted effective packaging of several types of RNAs and enabled efficient transfer of biologically active RNAs in various cell types, including human CD34(+) and iPS cells. Systemic injection of high-titer particles led to gene expression in mouse liver and transferring Cre-recombinase mRNA in muscle permitted widespread editing at the ROSA26 locus. We could further show that the VLPs were able to activate an osteoblast differentiation pathway by delivering RUNX2- or DLX5-mRNA into primary human bone-marrow mesenchymal-stem cells. Thus, the novel chimeric MS2-lentiviral particles are a versatile tool for a wide range of applications including cellular-programming or genome-editing.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: A previously published clinical trial demonstrated the benefit of autologous CD34+ cells transduced with a self-inactivating lentiviral vector (HPV569) containing an engineered β-globin gene (βA-T87Q globin) in a subject with β-thalassemia major. This vector has been modified to increase transduction efficacy without compromising safety. In vitro analyses indicated that the changes resulted in both increased vector titers (3 to 4 fold) and increased transduction efficacy (2 to 3 fold). An in vivo study in which 58 β-thalassemic mice were transplanted with vector- or mock-transduced syngenic bone marrow cells indicated sustained therapeutic efficacy. Secondary transplantations involving 108 recipients were performed to evaluate long-term safety. The six month study showed no hematological or biochemical toxicity. Integration site (IS) profile revealed an oligo/polyclonal hematopoietic reconstitution in the primary transplants and reduced clonality in secondary transplants. Tumor cells were detected in the secondary transplant mice in all treatment groups (including the control group), without statistical differences in the tumor incidence. Immunohistochemistry and quantitative PCR demonstrated that tumor cells were not derived from transduced donor cells. This comprehensive efficacy and safety data provided the basis for initiating two clinical trials with this second generation vector (BB305) in Europe and in the USA in patients with β-thalassemia major and sickle cell disease.
Current Gene Therapy 11/2014; 15(1). DOI:10.2174/1566523214666141127095336 · 2.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: iPSC-based therapy is hardly conceivable in humans without prior validation in large preclinical animal models closely related to humans. We generated iPSCs from Macaca cynomolgus to evaluate the engraftment capacity of cy-iPSC-derived hematopoietic cells after autologous transplantation. We first optimized a hematopoietic differentiation protocol based on an EB approach and followed the differentiation process over 3 weeks by time-lapse FACS and CFC assays to identify emerging cells with hemangioblastic and hematopoietic phenotypes. Up to 20% of CD34 + cells emerged around Day 12 to 15 of differentiation; however, fewer than 1/200 CD34 + cells were able to form CFCs. We analyzed the expression of genes associated with mesodermal and hemato/endothelial differentiation during the 3 weeks of differentiation. Expression of hematopoietic genes was consistent with the emergence of hematopoietic cells as determined by FACS and CFC assays. To evaluate the engraftment potential of cy-iPSC-derived hematopoietic cells, we injected 106 cells into either the femur (12 mice) or the retroorbital sinus (10 mice) of sublethally irradiated NSG mice. At 5–7 weeks post-injection, hematopoietic engraftment was observed in 9/12 mice that received an intra-femoral injection and in none of the mice that received a retro-orbital injection. Up to 0.5% of mouse BM cells stained positive for a macaca CD45 + antibody. CD45 +CD14 + , CD45 +CD11b + myeloid and CD45 + CD20 + lymphoid macaca populations were detected. Large-scale production of hematopoietic cells is currently underway to enable the autologous transplantation of cells to donor monkeys in order to evaluate the capacity of these cells for short and long-term hematopoietic engraftment.
ESGCT and NVGCT Collaborative Congress: The Hague-23 to 26 October; 10/2014
[Show abstract][Hide abstract] ABSTRACT: β-Thalassaemia major (β-TM) is an inherited haemoglobinopathy caused by a quantitative defect in the synthesis of β-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 β-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 β-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 β-TM erythroblasts, which may provide a rationale for new targeted therapies of β-TM.
[Show abstract][Hide abstract] ABSTRACT: Gene therapy, cellular immunotherapy and vaccination
ABSSUB-3287 (abstract Number S742)
OUTCOMES OF GENE THERAPY FOR BETA-THALASSEMIA MAJOR VIA TRANSPLANTATION OF AUTOLOGOUS HEMATOPOIETIC STEM CELLS TRANSDUCED EX VIVO WITH A LENTIVIRAL BETA GLOBIN VECTOR.
Marina Cavazzana* 1, Jean-Antoine Ribeil^1, Emmanuel Payen^2, 3, Felipe Suarez4, Olivier Negre5, Yves Beuzard2, 3, Fabien Touzot1, Resy Cavallesco6, François Lefrere1, Stany Chretien2, 3, Philippe Bourget7, Fabrice Monpoux8, Corinne Pondarre9, Benedicte Neven10, Frederic D. Bushman11, Manfred Schmidt12, Christof von Kalle12, Laura Sandler13, Sandeep Soni13, Byoung Ryu13, Robert Kutner13, Gabor Veres13, Mitchell Finer13, Stéphane Blanche10, Olivier Hermine4, Salima Hacein-Bey-Abina1, Philippe Leboulch2, 3, 6
1Département de Biothérapie, Hôpital Universitaire Necker - Enfants Malades, Paris, 2CEA, Institute of Emerging Diseases and Innovative Therapies (iMETI), 3UMR 962 (Inserm-CEA-University of Paris-Sud), Fontenay-aux-Roses, 4Service d'hématologie Adulte, Hôpital Universitaire Necker - Enfants Malades, Paris, 5bluebird bio France, CEA-iMETI, Fontenay-aux-Roses, France, 6Harvard Medical School and Brigham & Women's Hospital, Boston, United States, 7Clinical Pharmacy Department, Hôpital Universitaire Necker - Enfants Malades, Paris, 8Unité d’Hemato-Oncologie infantile, Centre Hospitalier de Nice Sophia-Antipolis, Nice, 9Service de pédiatrie, Centre de référence de la Drépanocytose, Centre Hospitalier Intercommunal de Créteil (CHIC), Créteil, 10Unité d'Immunologie-Hématologie Pédiatrique, Hôpital Universitaire Necker - Enfants Malades, Paris, France, 11Department of Microbiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, United States, 12Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany, 13bluebird bio, Cambridge, United States
Background: In patients with β-thalassemia major, haematopoietic stem cell (HSC) gene therapy has the potential to induce production of β-globin, γ-globin or modified β-globin in the RBC lineage and reduce or stop the need for transfusions. A prior clinical trial (LG001) demonstrated benefit of autologous CD34+ cells transduced with a replication-defective, self-inactivating lentiviral vector (HPV569) containing an engineered β-globin gene (βA-T87Q). A further modified βA-T87Q vector (LentiGlobin BB305) has achieved greater transduction efficiency and a similar pre-clinical safety profile. LentiGlobin BB305 is now being evaluated in the HGB-205 clinical trial.
Aims: To provide (i) long-term follow-up data on two subjects treated in LG001 and (ii) initial results from the HGB-205 study.
Methods: After the provision of informed consent, subjects with β-thalassemia major underwent HSC collection via peripheral blood apheresis and CD34+ cells were selected. Estimation of the mean ex vivo vector copy number (VCN) was obtained by qPCR performed on pooled in vitro colony-forming progenitors. Subjects underwent myeloablation with intravenous busulfan, followed by infusion of transduced CD34+ cells. Subjects were monitored for haematological engraftment, βA-T87Q expression (by HPLC) and transfusion requirements. Integration site analysis (by LAM-PCR and high-throughput sequencing on nucleated cells) and replication-competent lentivirus assays were performed.
Results: In LG001, two subjects (#1003 and #1004) with βE/β0 thalassemia major successfully engrafted following gene therapy with autologous HSCs transduced with HPV569. Neither subject experienced a cell infusion related adverse event. As reported previously (Nature 2010), #1003 became transfusion-independent one year post–transplant and remains so 5 years later, with the production of 2.5 - 3.5 g/dL βA-T87Q-globin (~30% of total haemoglobin). The most recent VCN in #1003's peripheral neutrophils is 0.23. Subject #1003 also demonstrated partial dominance of a clone with vector integration within the HMGA2 gene that peaked at 4 years post-treatment (22% of the nucleated cells) and has now fallen to 6.8% while maintaining transfusion independence. For #1004, the current VCN (2 years post-treatment) in neutrophils is 0.016 and βA-T87Q–globin accounts for ~5% of total haemoglobin. This subject remains transfusion dependent.
Two subjects with βE/β0 thalassemia major (#1201 and #1202) have enrolled in the current HGB-205 trial and one has undergone transplantation. Transduction efficiency of the new BB305 vector compared to HPV569 is shown in Table 1. Data on the transplant outcomes and up to 6 months of follow-up in subjects treated in the HGB-205 trial will be presented at the meeting.
Summary/Conclusion: Long-term transfusion independence is achievable with gene therapy for β thalassemia major. Use of the LentiGlobin BB305 vector has resulted in substantially higher VCN in CD34+ cells. It remains to be seen how the clinical outcomes will reflect this improvement.
Table1. Comparison of gene transfer efficiencies and transplantation outcomes with HPV569 vs. BB305 βA‑T87Q‑LentiGlobin vectors
^these authors contributed equally
Keywords: beta thalassemia, Gene therapy, Lentiviral vector, Transduction
European Hematology Association - 19th Congress, Milano (Italy); 06/2014
[Show abstract][Hide abstract] ABSTRACT: The pathophysiology of ineffective erythropoiesis in β-thalassemia is poorly understood. We report that RAP-011, an activin receptor IIA (ActRIIA) ligand trap, improved ineffective erythropoiesis, corrected anemia and limited iron overload in a mouse model of β-thalassemia intermedia. Expression of growth differentiation factor 11 (GDF11), an ActRIIA ligand, was increased in splenic erythroblasts from thalassemic mice and in erythroblasts and sera from subjects with β-thalassemia. Inactivation of GDF11 decreased oxidative stress and the amount of α-globin membrane precipitates, resulting in increased terminal erythroid differentiation. Abnormal GDF11 expression was dependent on reactive oxygen species, suggesting the existence of an autocrine amplification loop in β-thalassemia. GDF11 inactivation also corrected the abnormal ratio of immature/mature erythroblasts by inducing apoptosis of immature erythroblasts through the Fas-Fas ligand pathway. Taken together, these observations suggest that ActRIIA ligand traps may have therapeutic relevance in β-thalassemia by suppressing the deleterious effects of GDF11, a cytokine which blocks terminal erythroid maturation through an autocrine amplification loop involving oxidative stress and α-globin precipitation.
Nature medicine 03/2014; 20(4). DOI:10.1038/nm.3468 · 27.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Our understanding of system dynamics of mixed cell populations in whole organisms has benefited from the advent of individual cell marking by non-arrayed 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.
[Show abstract][Hide abstract] ABSTRACT: A patient with β(β) /β(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, β-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 mice, embryonic to fetal and a partial fetal to adult globin class switching were observed, indicating that gene transfer is likely necessary for iPSC-based therapy of the β-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 non-isogenic gene therapy patients with β-thalassemia and adrenoleukodystrophy, respectively. This suggests that CIS observed in the absence of overt tumorigenesis result from non-random 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.
[Show abstract][Hide abstract] ABSTRACT: High-level production of β-globin, γ-globin, or therapeutic mutant globins in the RBC lineage by hematopoietic stem cell gene therapy ameliorates or cures the hemoglobinopathies sickle cell disease and beta thalassemia, which are major causes of morbidity and mortality worldwide. Considerable efforts have been made in the last 2 decades in devising suitable gene-transfer vectors and protocols to achieve this goal. Five years ago, the first β(E)/β(0)-thalassemia major (transfusion-dependent) patient was treated by globin lentiviral gene therapy without injection of backup cells. This patient has become completely transfusion independent for the past 4 years and has global amelioration of the thalassemic phenotype. Partial clonal dominance for an intragenic site (HMGA2) of chromosomal integration of the vector was observed in this patient without a loss of hematopoietic homeostasis. Other patients are now receiving transplantations while researchers are carefully weighing the benefit/risk ratio and continuing the development of further modified vectors and protocols to improve outcomes further with respect to safety and efficacy.
[Show abstract][Hide abstract] ABSTRACT: Malignant transformation is a multistep process requiring oncogenic activation, promoting cellular proliferation, frequently coupled to inhibition of terminal differentiation. Consequently, forcing the reengagement of terminal differentiation of transformed cells coupled or not with an inhibition of their proliferation is a putative therapeutic approach to counteracting tumorigenicity. UT7 is a human leukemic cell line able to grow in the presence of IL3, GM-CSF and Epo. This cell line has been widely used to study Epo-R/Epo signaling pathways but is a poor model for erythroid differentiation. We used the BET bromodomain inhibition drug JQ1 to target gene expression, including that of c-Myc. We shown that only two days of JQ1 treatment was required to transitory inhibit Epo-induced UT7 proliferation and to restore terminal erythroid differentiation. This study highlights the importance of a cellular erythroid cycle break mediated by c-Myc inhibition before initiation of the erythropoiesis program and describes a new model for BET bromodomain inhibitor drug application.
Biochemical and Biophysical Research Communications 11/2012; 429(1-2). DOI:10.1016/j.bbrc.2012.10.112 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Integration of retroviral DNA into host cell DNA is a defining feature of retroviral replication. The integration system is important as a new a drug target, as a model for function of molecular machines that change genomes, and as a tool for use in human gene therapy. The lecture will cover the mechanisms underlying integration and the consequences for the host cell. One line of work to be discussed centers on the mechanisms by which retroviruses select integration target sites in chromosomes. HIV integration is known to be favored in active transcription units, which promotes efficient transcription of the viral genes. A host cell factor, PSIP1/LEDGF/p75 acts as a tethering factor to direct local integration, accounting for part of the effect. In addition, the methods devised for basic studies of retroviral integration targeting are useful for analyzing integration sites generated during human gene therapy. Selected results from such studies will also be discussed.
[Show abstract][Hide abstract] ABSTRACT: Although clinical benefits have been reported in several human hematopoietic gene therapy trials, a remaining important goal is the transition to nonmyeloablative pretransplantation conditioning to decrease toxicity. Previous attempts at reduced intensity conditioning in nonhuman primates have resulted in only temporary vector marking of autologous blood cells or their persistence at low levels, well below the thresholds for clinical efficacy. In addition, we reasoned that lentiviral vector particles displaying cytokines at their surface have the potential to preserve stem cell fitness better than current ex vivo transduction protocols, which involve exposure to cytokine overstimulation. Here we show that the classically nonmyeloablative agent fludarabine (30 mg/m(2)/day for 3 days) together with low-level total body irradiation (2 Gy) and the use of a stem cell factor-displaying simian immunodeficiency virus-based vector, resulted in sustained, single-copy vector marking of autologous blood cells in two macaques over 3 years posttransplantation at levels averaging 1% of all lineages. This percentage is within the range of anticipated efficacy levels for hemophilia and related diseases and forms a basis for further improvement.
Human gene therapy 03/2012; 23(7):754-68. DOI:10.1089/hum.2012.020 · 3.76 Impact Factor