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A primary challenge in lentiviral gene therapy of β-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, LVβ-shα2, that allows coordinated expression of the therapeutic βA-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 β-thalassemia. We demonstrate that LVβ-shα2 reduces α2-globin mRNA expression in erythroid cells while keeping α1-globin mRNA levels unchanged and βA-T87Q-globin gene expression identical to the parent vector. Compared with the first βA-T87Q-globin lentiviral vector that has received conditional marketing authorization, BB305, LVβ-shα2 shows 1.7-fold greater potency to improve α/β ratios. It may thus result in greater therapeutic efficacy and reliability for the most severe types of β-thalassemia and provide an improved benefit/risk ratio regardless of the β-thalassemia genotype.

Recent marketing approval for genetically engineered hematopoietic stem and T cells bears witness to the substantial improvements in lentiviral vectors over the last two decades, but evaluations of...

Sickle cell disease (SCD) and transfusion-dependent β-thalassemia (TDT) are the most prevalent monogenic disorders worldwide. Trial HGB-205 ( NCT02151526 ) aimed at evaluating gene therapy by autologous CD34

The β-hemoglobinopathies, transfusion-dependent β-thalassemia and sickle cell disease, are the most prevalent inherited disorders worldwide and affect millions of people. Many of these patients have a shortened life expectancy and suffer from severe morbidity despite supportive therapies, which impose an enormous financial burden to societies. The only available curative therapy is allogeneic hematopoietic stem cell transplantation, although most patients do not have an HLA-matched sibling donor, and those who do still risk life-threatening complications. Therefore, gene therapy by one-time ex vivo modification of hematopoietic stem cells followed by autologous engraftment is an attractive new therapeutic modality. The first proof-of-principle of conversion to transfusion independence by means of a lentiviral vector expressing a marked and anti-sickling βT87Q-globin gene variant was reported a decade ago in a patient with transfusion-dependent β-thalassemia. In follow-up multicenter Phase II trials with an essentially identical vector (termed LentiGlobin BB305) and protocol, 12 of the 13 patients with a non-β0/β0 genotype, representing more than half of all transfusion-dependent β-thalassemia cases worldwide, stopped red blood cell transfusions with total hemoglobin levels in blood approaching normal values. Correction of biological markers of dyserythropoiesis was achieved in evaluated patients. In nine patients with β0/β0 transfusion-dependent β-thalassemia or equivalent severity (βIVS1-110), median annualized transfusion volume decreased by 73% and red blood cell transfusions were stopped in three patients. Proof-of-principle of therapeutic efficacy in the first patient with sickle cell disease was also reported with LentiGlobin BB305. Encouraging results were presented in children with transfusion-dependent β-thalassemia in another trial with the GLOBE lentiviral vector and several other gene therapy trials are currently open for both transfusion-dependent β-thalassemia and sickle cell disease. Phase III trials are now under way and should help to determine benefit/risk/cost ratios to move gene therapy toward clinical practice.

Recent marketing approval for genetically engineered hematopoietic stem and T cells bears witness to the substantial improvements in lentiviral vectors over the last two decades, but evaluations of the long-term efficacy and toxicity of gene and cell therapy products will, nevertheless, require further studies in nonhuman primate models.

β-globin gene disorders are the most prevalent inherited diseases worldwide and result from abnormal β-globin synthesis or structure. Novel therapeutic approaches are being developed in an effort to move beyond palliative management. Gene therapy, by ex vivo lentiviral transfer of a therapeutic β-globin gene derivative (β(AT87Q)-globin) to hematopoietic stem cells, driven by cis-regulatory elements that confer high, erythroid-specific expression, has been evaluated in human clinical trials over the past 8 years. β(AT87Q)-globin is used both as a strong inhibitor of HbS polymerization and as a biomarker. While long-term studies are underway in multiple centers in Europe and in the United States, proof-of-principle of efficacy and safety has already been obtained in multiple patients with β-thalassemia and sickle cell disease.

Although gene transfer to hematopoietic stem cells (HSCs) has shown therapeutic efficacy in recent trials for several individuals with inherited disorders, transduction incompleteness of the HSC population remains a hurdle to yield a cure for all patients with reasonably low integrated vector numbers. In previous attempts at HSC selection, massive loss of transduced HSCs, contamination with non-transduced cells, or lack of applicability to large cell populations has rendered the procedures out of reach for human applications. Here, we fused codon-optimized puromycin N-acetyltransferase to herpes simplex virus thymidine kinase. When expressed from a ubiquitous promoter within a complex lentiviral vector comprising the βAT87Q-globin gene, viral titers and therapeutic gene expression were maintained at effective levels. Complete selection and preservation of transduced HSCs were achieved after brief exposure to puromycin in the presence of MDR1 blocking agents, suggesting the procedure’s suitability for human clinical applications while affording the additional safety of conditional suicide., Recent clinical trials have demonstrated the benefits of hematopoietic gene therapy using lentiviral vectors. In this paper, Payen and colleagues describe a method to maximize the proportion of genetically modified human hematopoietic stem cells while limiting the mean vector copy number.

International audience; BACKGROUND Donor availability and transplantation-related risks limit the broad use of allogeneic hematopoietic-cell transplantation in patients with transfusion-dependent beta-thalassemia. After previously establishing that lentiviral transfer of a marked beta-globin (beta(A-T87Q)) gene could substitute for long-term red-cell transfusions in a patient with beta-thalassemia, we wanted to evaluate the safety and efficacy of such gene therapy in patients with transfusion-dependent beta-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 beta-thalassemia and transduced the cells ex vivo with LentiGlobin BB305 vector, which encodes adult hemoglobin (HbA) with a T87Q amino acid substitution (HbA(T87Q)). 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 HbA(T87Q), 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-beta(0)/beta(0) genotype had stopped receiving red-cell transfusions; the levels of HbA(T87Q) 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 beta(0)/beta(0) 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 beta-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.)

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 .).

A previously published clinical trial demonstrated the benefit of autologous CD34 + cells transduced with a selfinactivating lentiviral vector (HPV569) containing an engineered β-globin gene (β A-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.

The very large number of known mutations responsible for the β-thalassemias, renders gene therapy by gene addition ideally suited for regulatory product approval rather than the many mutation-speci...

A patient with βE/β0-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 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 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 nonisogenic gene therapy patients with β-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.

A lentiviral vector encoding β-globin flanked by insulator elements has been used to treat β-thalassemia (β-Thal) successfully in one human subject. However, a clonal expansion was observed after integration in the HMGA2 locus, raising the question of how commonly lentiviral integration would be associated with possible insertional activation. Here, we report correcting β-Thal in a murine model using the same vector and a busulfan-conditioning regimen, allowing us to investigate efficacy and clonal evolution at 9.2 months after transplantation of bone marrow cells. The five gene-corrected recipient mice showed near normal levels of hemoglobin, reduced accumulation of reticulocytes, and normalization of spleen weights. Mapping of integration sites pretransplantation showed the expected favored integration in transcription units. The numbers of gene-corrected long-term repopulating cells deduced from the numbers of unique integrants indicated oligoclonal reconstitution. Clonal abundance was quantified using a Mu transposon-mediated method, indicating that clones with integration sites near growth-control genes were not enriched during growth. No integration sites involving HMGA2 were detected. Cells containing integration sites in genes became less common after prolonged growth, suggesting negative selection. Thus, β-Thal gene correction in mice can be achieved without expansion of cells harboring vectors integrated near genes involved in growth control.

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 β-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 β-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.

Objective To investigate whether expression of a membrane-anchored form of erythropoietin (MbEpo) results in self-controlled, autocrine proliferation, and differentiation of erythroid cells. This would provide a possible approach to the selective expansion of genetically corrected erythroid cells in gene-therapy protocols. Materials and Methods We designed retroviral vectors encoding MbEpo or secreted erythropoietin (Epo) and enhanced green fluorescent protein. Several Epo-dependent cell lines were transduced and their proliferative capacity evaluated. This approach was also assessed in human bone marrow CD34 + cells and mouse bone marrow transplants. Results Retroviral vector–mediated MbEpo expression induced autocrine proliferation of the Epo-dependent cell lines DAE7 and UT7/Epo. However, it blocked the Epo receptor (EpoR)–induced activation of granulocyte macrophage colony-stimulating factor–dependent UT7/GM cells and the erythroid differentiation of both human hematopoietic cells in vitro and of mouse bone marrow cells in transplant experiments. MbEpo was present at the surface of UT7/GM cells. It did not affect the membrane localization of the EpoR, but prevented its normal Epo-dependent phosphorylation and internalization. By contrast to these inhibitory effects, a higher rate of EpoR replenishment in UT7/GM cells before MbEpo production rendered cell proliferation independent of exogenous growth factor. Conclusions Activation of EpoR gene expression before MbEpo-induced EpoR activation is essential for activation or inhibition of growth and differentiation of Epo-dependent cell lines. It will be necessary to delay MbEpo expression in late erythroid progenitors until after EpoR gene activation, for erythroid cell expansion to be achieved in vivo.

We first reported that of the 10 most commonly used AAV serotype vectors, AAV6 is the most efficient in transducing primary human bone marrow-derived CD34+ hematopoietic stem/progenitor cells (HSPCs), both in vitro and in murine xenograft models in vivo (Cytotherapy, 15: 986-998, 2013; PLoS One, 8(3): e58757, 2013). More recently, two independent groups also reported successful transduction of primary human CD34+ cells using the wild-type (WT) AAV6 vectors (Sci. Transl. Med., 7: 307ra156, 2015; Nat. Biotechnol., 33: 1256-1263, 2015), except that multiplicities of infection (MOIs) ranging from 100,000-200,000 vgs/cell were used to achieve ~40-55% transduction efficiency. Furthermore, the transduction efficiency of the WT AAV6 vector varies greatly in HSPCs from different donors, ranging between ~6-87%. Here we report two distinct strategies to further increase the transduction efficiency in HSPCs from donors that are transduced poorly with the WT AAV6 vectors. The first strategy involved modification of the viral capsid proteins where specific surface-exposed tyrosine (Y) and threonine (T) residues were mutagenized to generate a triple-mutant (Y705F+Y731F+T491V) AAV6 vector. The second strategy involved the use of ex vivo transduction at high cell density, which revealed a novel mechanism, which we have termed, ‘cross-transduction’. The combined use of these strategies resulted in transduction efficiency exceeding 90% at an MOI of 20,000 vgs/cell in primary human cord blood-derived HSPCs at day 4 (Fig. 1AFig. 1A). scAAV6 vectors were more efficient than ssAAV6 vectors, but at high cell density, there was a modest enhancement in EGFP-positivity even with ssAAV6 vectors. However, 14 days post-transduction, virtually no EGFP-positive cells could be detected (Fig. 1BFig. 1B), suggesting the loss of vector genomes, and hence, the lack of stable integration of vector genomes in HSPCs. Our studies have significant implications in the optimal use of capsid-optimized AAV6 vectors in genome editing in HSPCs. *These authors contributed equally to this work #Co-corresponding authorsView Large Image | Download PowerPoint Slide

Achieving long-term pancellular expression of a transferred gene at therapeutic level in a given hematopoietic lineage remains an important goal of gene therapy. Advances have recently been made in the genetic correction of the hemoglobinopathies by means of lentiviral vectors and large locus control region (LCR) derivatives. However, panerythroid beta globin gene expression has not yet been achieved in beta thalassemic mice because of incomplete transduction of the hematopoietic stem cell compartment and position effect variegation of proviruses integrated at a single copy per genome. Here, we report the permanent, panerythroid correction of severe beta thalassemia in mice, resulting from a homozygous deletion of the beta major globin gene, by transplantation of syngeneic bone marrow transduced with an HIV-1-derived [beta globin gene/LCR] lentiviral vector also containing the Rev responsive element and the central polypurine tract/DNA flap. The viral titers produced were high enough to achieve transduction of virtually all of the hematopoietic stem cells in the graft with an average of three integrated proviral copies per genome in all transplanted mice; the transduction was sustained for7 months in both primary and secondary transplants, at which time approximately 95% of the red blood cells in all mice contained human beta globin contributing to 32 +/- 4% of all beta-like globin chains. Hematological parameters approached complete phenotypic correction, as assessed by hemoglobin levels and reticulocyte and red blood cell counts. All circulating red blood cells became and remained normocytic and normochromic, and their density was normalized. Free alpha globin chains were completely cleared from red blood cell membranes, splenomegaly abated, and iron deposit was almost eliminated in liver sections. These findings indicate that virtually complete transduction of the hematopoietic stem cell compartment can be achieved by high-titer lentiviral vectors and that position effect variegation can be mitigated by multiple events of proviral integration to yield balanced, panerythroid expression. These results provide a solid foundation for the initiation of human clinical trials in beta thalassemia patients.

Sickle cell disease (SCD) is caused by a single point mutation in the human β A globin gene that results in the formation of an abnormal hemoglobin [HbS (α 2 β S 2 )]. We designed a β A globin gene variant that prevents HbS polymerization and introduced it into a lentiviral vector we optimized for transfer to hematopoietic stem cells and gene expression in the adult red blood cell lineage. Long-term expression (up to 10 months) was achieved, without preselection, in all transplanted mice with erythroid-specific accumulation of the antisickling protein in up to 52% of total hemoglobin and 99% of circulating red blood cells. In two mouse SCD models, Berkeley and SAD, inhibition of red blood cell dehydration and sickling was achieved with correction of hematological parameters, splenomegaly, and prevention of the characteristic urine concentration defect.

Objective A new intramuscular DNA electrotransfer method for erythropoietin (EPO) expression was evaluated in the natural mouse model of human β-thalassemia (Hbb-thal1) in terms of its ability to reverse the anemia and improve the thalassemic features of erythrocytes. Materials and Methods Intramuscular injection of small amounts of a plasmid encoding mouse EPO, immediately followed by controlled electric pulses, was used. Results This procedure induced very high hematocrit levels in β-thalassemic mice compared to nonelectrotransferred mice. The hematocrit increase was dose dependent, still increased 4 months after injection of plasmid DNA, and associated with a high transgenic EPO blood level in all mice (up to 2500 mU/mL of plasma). EPO gene electrotransfer not only led to a long-lasting and dose-dependent increase in the hematocrit but also to a 100% increase in the lifespan of erythrocytes of thalassemic mice. This was related to a nearly complete reestablishment of α/β globin chain balance, as demonstrated by a marked decrease in unpaired α globin chain. Eight months after the first electrotransfer of pCMV-mEPO plasmid, reinjection of the same construct raised the hematocrit to a level close to that observed following the first electrotransfer. Conclusion This is the first description of the use of plasmid DNA to achieve long-term improvement in a mouse model of a human genetic disorder.

Background The combination of physiologically and pharmacologically controlled elements may provide a means to ensure both the regulation and the safety of transgene expression – two major goals in gene therapy. Methods A two-gene modulation system was developed that uses the following three levels of control: (i) the hypoxia-responsive element directing the transcription of the tetracycline-controlled transactivator (tTA); (ii) part of the oxygen-degradation domain limiting the production of tTA in normoxia; and (iii) the tetracycline switch of the transactivator activity (the tet-off system). Results This triple-control system allowed high expression of the gene of interest (luciferase or erythropoietin) by transfected cells upon hypoxia and low expression under normoxia or in the presence of tetracycline. This control of transgene expression was also obtained in mouse tumors. Conclusions This multiple-control system is of interest for spatially restricting transgene expression into hypoxic tumors, and for finely adjusting the expression level of a therapeutic protein to the oxygen supply in medical applications such as neoangiogenesis or the erythropoietin-mediated treatment of anemia. Copyright © 2001 John Wiley & Sons, Ltd.

Objective Future prospects for gene therapy of chronic anemias involve expression of the erythropoietin transgene, which is regulated by oxygen tension. However, other factors such as cytokines or the iron load of erythropoietin-expressing cells can concomitantly modulate transgene expression, as shown for the expression of the endogenous erythropoietin gene in human cell lines and in animals. We tested the effects of iron overload or depletion on the expression of the mouse erythropoietin transgene (cDNA), driven by the hypoxia-regulated phosphoglycerate kinase 1 promoter. Materials and Methods Retrovirally transduced mouse cells (C3H fibroblasts or C2C12 myoblasts) were cultured in normoxia (room air, O 2 : 21%) or hypoxia (O 2 : 1.5%) in the presence or absence of hemin (an iron donor) or deferiprone (an iron chelator), both of which easily enter the cell. Results Hemin inhibited the hypoxia-induced expression of the transgene. In contrast, deferiprone enhanced the hypoxia-induced expression of the erythropoietin transgene and induced its expression in normoxia. Conclusion These results show that, in addition to oxygen partial pressure, the intracellular iron content is critical in the modulation of hypoxia-regulated erythropoietin transgene expression.

Twenty-four hamster-sheep hybrid cell lines representing eleven ovine synteny groups were used to make syntenic assignments for seven loci ALDOB (aldolase B, fructose biophosphate); AMH (anti-Müllerian hormone); CYP19 [cytochrome P450 aromatase, subfamily XIX (aromatization of androgens)]; WT (Wilms' tumour gene); SOX2 (SRY-related HMG-box gene 2); FSHB (follicle-stimulating hormone, beta polypeptide); and SRY (sex region of Y chromosome). These loci were assigned to synteny groups U11(chr2) (ALDOB); U19 (AMH); U3(chr7) (CYP19); and to chromosome 15 (WT) and 1 (SOX2). SRY defines the hybrids containing the Y chromosome.

The SOX gene family consists of a large number of embryonically expressed genes capable of encoding putative transcription factors and related by a DNA-binding domain, the HMG-box. We cloned and characterized the ovine SOX2 transcript using the screening of a testis (12dpp) cDNA library with a probe containing the SRY-HMG-box and we performed 3'RACE experiments. The ovine SOX2 sequence is strongly conserved in comparison to the human, mouse and chicken homologues and is located on sheep chromosome 1q33. The SOX2 expression pattern in developing gonads is consistent with the hypothesis that this gene plays a role in the germ cell line.

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.

Because male ovine embryos develop faster than female embryos, the transcription of SRY and ZFY, two genes located on the Y chromosome, was examined in preimplantation stages using the reverse transcriptase polymerase chain reaction (RT-PCR). RNA was extracted from pools of ovine embryos matured and fertilized in vitro then cultured in synthetic oviduct fluid medium and recovered from 24 to 207 hr post-insemination (two-cell up to hatched blastocyst stage). Since primers used to amplify ZFY also amplify the homologue ZFX, located on the X chromosome, transcripts were differentiated by digestion with restriction enzymes. ZFY and ZFX transcripts were present in all stages examined following RT-PCR, whereas transcripts for SRY were undetectable in all investigated stages following either RT nested PCR or Southern analysis. The presence of ZFY transcripts suggests that Y chromosome is transcriptionally active during early ovine preimplantation development. The possible relationship between a faster growth of male embryos and the transcription of Y-linked genes at early stages of development is discussed.

We have reported that of the 10 commonly used AAV serotype vectors, AAV6 is the most efficient in transducing primary human hematopoietic stem/progenitor cells (HSPCs). However, the transduction efficiency of the wild-type (WT) AAV6 vector varies greatly in HSPCs from different donors. Here we report two distinct strategies to further increase the transduction efficiency in HSPCs from donors that are transduced poorly with the WT AAV6 vectors. The first strategy involved modification of the viral capsid proteins where specific surface-exposed tyrosine (Y) and threonine (T) residues were mutagenized to generate a triple-mutant (Y705+Y731F+T492V) AAV6 vector. The second strategy involved the use of ex vivo transduction at high cell density, which revealed a novel mechanism, which we have termed, 'cross-transduction'. The combined use of these strategies resulted in transduction efficiency exceeding ~90% in HSPCs. Our studies have significant implications in the optimal use of capsid-optimized AAV6 vectors in genome editing in HSPCs. Disclosures Leboulch: bluebird bio: Patents & Royalties. Payen:bluebird bio: Patents & Royalties. Srivastava:AGTC; Genzyme: Patents & Royalties.

Introduction: β-globin gene transfer into hematopoietic stem cells (HSCs) has the potential to reduce or eliminate the symptoms of severe sickle cell disease (SCD) and reduce or eliminate transfusion requirements in transfusion-dependent β-thalassemia (TDT). LentiGlobin Drug Product (DP) contains autologous CD34+ cells transduced with the BB305 lentiviral vector, which encodes a human β-globin gene containing a single point mutation (AT87Q) designed to confer anti-sickling properties similar to those observed with γ-globin. We previously reported proof of concept for LentiGlobin DP treatment in severe SCD and early data from 4 treated patients with TDT. We now report 18 months of follow-up for the patient with SCD and between 9 and 30 months of follow-up for the 4 patients with TDT. Patients (5-35 years of age) with severe SCD (e.g. ≥2 acute chest syndrome episodes or ≥2 vaso-occlusive crises [VOC] in preceding year/in year prior to regular transfusions) or TDT (≥100mL/kg of packed red blood cells [RBCs] per year) were enrolled. Following mobilization and apheresis (for TDT) or bone marrow harvest (for SCD), autologous CD34+ cells were transduced with the BB305 lentiviral vector. Patients underwent myeloablative conditioning with busulfan prior to infusion of the transduced cells. After infusion, patients were monitored for hematologic engraftment, vector copy number (VCN), and HbAT87Q expression. Disease-specific assessments included transfusion requirements for TDT, or VOCs and hospitalizations for SCD. Safety assessments included adverse events (AEs) and integration site analysis. Results: As of July 2016, 1 patient with severe SCD (male; 13 years old) and 4 patients with TDT (2 male, 2 female; 16-19 years old) have received LentiGlobin DP in Study HGB-205. The median LentiGlobin DP cell dose was 8.9 (range 5.6-13.6) x 106 CD34+ cells/kg with a DP VCN of 1.2 (range: 0.8-2.0) vector copies/diploid genome. Median post-infusion follow-up as of July 6, 2016 is 20.8 months (range 9.5-31.3). All subjects successfully engrafted after receiving LentiGlobin DP, with a median time to neutrophil engraftment of 17 days (range 14-38 days). VCN in peripheral blood has remained generally consistent from Month 3 in all patients with a range of 0.2 to 3.4 at last measurement. The toxicity profile observed from start of conditioning to latest follow-up remains consistent with myeloablative conditioning with single-agent busulfan, with no DP-related ≥Grade 3 AEs or serious AEs and no evidence of clonal dominance reported to date. Three patients with TDT have β0/βE genotypes and 1 is homozygous for the severe β+ mutation IVS1 nt 110 G>A. The 2 patients who have completed the 2-year primary follow-up period (both β0/βE) have not required RBC transfusions for 31 and 28 months, with total Hb of 10.9 and 13.5 g/dL, and HbAT87Q expression of 7.7 and 10.1 g/dL, respectively, at most recent study visit. Iron chelation has been discontinued and phlebotomy initiated for 1 of the patients. The remaining patient with β0/βE genotype has 9 months of follow-up and has not required transfusions since 4 days post-LentiGlobin DP infusion, achieving a total Hb of 11.3 g/dL at last study visit. The patient with the severe IVS1 genotype has 12 months of follow-up and has been free of transfusions for 9 months, with a total Hb of 8.3 g/dL at last study visit. The patient with severe SCD, who prior to study enrollment received regular RBC transfusions, has experienced no clinical symptoms or complications of SCD in the 18 months since treatment, despite discontinuing transfusions 3 months after LentiGlobin DP infusion. Total Hb in this patient was 12.5 g/dL, with 6.6 g/dL HbAT87Q (53%) and 5.7 g/dL HbS (45%) at last study visit. Compared with values at screening, unconjugated bilirubin had dropped 78% (50 to 11 μmol/L), lactate dehydrogenase had dropped 54% (626 to 287 U/L), and reticulocyte count had dropped 45% (238x109/L to 132x109/L) by Month 18. Conclusions: Data from this ongoing Phase 1/2 clinical study suggest that treatment with LentiGlobin DP can result in sustained production of therapeutic HbAT87Q, which ameliorates the clinical and biochemical effects of severe SCD and TDT, with an acceptable safety profile. Gene therapy presents a potentially promising therapy for patients with severe hemoglobinopathies. Further follow-up and additional data from patients are needed to confirm the encouraging results seen to date in this study. Disclosures Ribeil: Bluebirdbio: Consultancy; Addmedica: Research Funding. Payen:bluebird bio: Patents & Royalties. Hermine:Alexion: Research Funding; Celgene: Research Funding; Novartis: Research Funding; AB science: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding, Speakers Bureau. Asmal:bluebird bio: Employment, Equity Ownership. Joseney-Antoine:bluebird bio: Employment, Equity Ownership. De Montalembert:Addmedica: Research Funding; Novartis: Research Funding, Speakers Bureau. Leboulch:bluebird bio, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Patients with β-thalassemia major require lifelong transfusions and iron chelation, regardless of the type of causative mutations (e.g., β⁰, β(E)/β⁰). The only available curative therapy is allogeneic hematopoietic transplantation, although most patients do not have an HLA-matched, geno-identical donor, and those who do still risk graft-versus-host disease. Hence, gene therapy by ex vivo transfer of a functional β-globin gene is an attractive novel therapeutic modality. In β-thalassemia, transfer of a therapeutic globin gene does not confer a selective advantage to transduced stem cells, and complex DNA regulatory sequences have to be present within the transfer vector for proper expression. This is why lentiviral vectors have proven especially suited for this application, and the first Phase I/II human clinical trial was initiated. Here, we report on the first gene therapy patient with severe β(E)/β⁰-thalassemia, who has become transfusion-independent, and provide methods and protocols used in the context of this clinical trial.

Patients with β-thalassemia major require lifelong transfusions and iron chelation, regardless of the type of causative mutations (e.g., β⁰, β(E)/β⁰). The only available curative therapy is allogeneic hematopoietic transplantation, although most patients do not have an HLA-matched, geno-identical donor, and those who do still risk graft-versus-host disease. Hence, gene therapy by ex vivo transfer of a functional β-globin gene is an attractive novel therapeutic modality. In β-thalassemia, transfer of a therapeutic globin gene does not confer a selective advantage to transduced stem cells, and complex DNA regulatory sequences have to be present within the transfer vector for proper expression. This is why lentiviral vectors have proven especially suited for this application, and the first Phase I/II human clinical trial was initiated. Here, we report on the first gene therapy patient with severe β(E)/β⁰-thalassemia, who has become transfusion-independent, and provide methods and protocols used in the context of this clinical trial.

Blood disorders caused by abnormal β-globin — β-thalassaemia and sickle cell disease — are the most prevalent inherited disorders worldwide, with patients often remaining dependent on blood transfusions throughout their lives. So a report of the successful use of gene therapy in a case of severe β-thalassaemia — using a lentiviral vector expressing the β-globin gene — is an eagerly awaited event. More than two years after gene transfer, the adult male patient has been transfusion-independent for 21 months. The therapeutic benefit seems to result from a dominant, myeloid-biased cell clone that may remain benign, although it could yet develop into leukaemia — a reminder that gene therapy is still at an early stage. Disorders caused by abnormal β-globin, such as β-thalassaemia, are the most prevalent inherited disorders worldwide. For treatment, many patients are dependent on blood transfusions; thus far the only cure has involved matched transplantation of haematopoietic stem cells. Here it is shown that lentiviral β-globin gene transfer can be an effective substitute for regular transfusions in a patient with severe β-thalassaemia. The β-haemoglobinopathies are the most prevalent inherited disorders worldwide. Gene therapy of β-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 βE/β0-thalassaemia is the most common form of severe thalassaemia in southeast Asian countries and their diasporas1,2. The βE-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 βE-globin with partial instability1,2. When this is compounded with a non-functional β0 allele, a profound decrease in β-globin synthesis results, and approximately half of βE/β0-thalassaemia patients are transfusion-dependent1,2. 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 β-globin gene transfer, an adult patient with severe βE/β0-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 β-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.

Slowing Brain Disease with Gene Therapy X-linked adrenoleukodystrophy (ALD), the hereditary brain demyelinating disorder that was featured in the movie “ Lorenzo's Oil ,” is typically treated by transplantation of bone marrow from matched donors. This treatment slows progression of the disease by introducing cells that differentiate into myelin-producing cells. Cartier et al. (p. 818 ; see Perspective by Naldini ) tested an alternative gene therapy–based approach in two young patients without matched donors. A lentiviral vector was used to introduce a wild-type copy of the ALD gene into the patients' hematopoietic stem cells ex vivo. The modified cells were then infused back into the patients. Expression of the transferred gene was still detectable in the patients' blood cells 2 years later, and both patients showed neurological improvement and a delay in disease progression comparable to that seen with bone marrow transplants.

Numerous studies using erythropoietin (EPO) gene delivery vectors, either viral or nonviral, have shown uncontrolled EPO expression leading to transient or sustained erythrocytosis and, more recently, severe autoimmune anemia. Therefore, there is a need to develop other EPO gene delivery systems that allow sustained and adjustable expression of EPO. We have examined a new approach of delivering plasmid encoding mouse EPO cDNA into mouse skeletal muscle, using an amphiphilic block copolymer. Repeated injections of low doses of block copolymer-EPOcDNA formulations increased hematocrit in a dose-dependent manner for more than 9 months, without any initial overshoot. Low doses of block copolymer-EPOcDNA formulations prevented autoimmune anemia in immunocompetent Swiss mice and prevented or reversed chronic anemia in an acquired mouse model of renal failure. We conclude that repeated injections of low doses of block copolymer-DNA formulations that do not induce (1) inflammation at the injection site, (2) overexpression of EPO, or (3) the production of anti-EPO neutralizing auto-antibodies hold promise for in vivo expression of therapeutic proteins, in particular for systemic delivery.

Background In situ production of a secreted therapeutic protein is one of the major gene therapy applications. Nevertheless, the plasmatic secretion peak of transgenic protein may be deleterious in many gene therapy applications including Epo gene therapy. Epo gene transfer appears to be a promising alternative to recombinant Epo therapy for severe anaemia treatment despite polycythemia was reached in many previous studies. Therefore, an accurate level of transgene expression is required for Epo application safety. The aim of this study was to adapt posology and administration schedule of a chosen therapeutic gene to avoid this potentially toxic plasmatic peak and maintain treatment efficiency. The therapeutic potential of repeated muscular electrotransfer of light Epo-plasmid doses was evaluated for anaemia treatment in β-thalassemic mice. Methods Muscular electrotransfer of 1 μg, 1.5 μg, 2 μg 4 μg or 6 μg of Epo-plasmid was performed in β-thalassemic mice. Electrotransfer was repeated first after 3.5 or 5 weeks first as a initiating dose and then according to hematocrit evolution. Results Muscular electrotransfer of the 1.5 μg Epo-plasmid dose repeated first after 5 weeks and then every 3 months was sufficient to restore a subnormal hematrocrit in β-thalassemic mice for more than 9 months. Conclusion This strategy led to efficient, long-lasting and non-toxic treatment of β-thalassemic mouse anaemia avoiding the deleterious initial hematocrit peak and maintaining a normal hematocrit with small fluctuation amplitude. This repeat delivery protocol of light doses of therapeutic gene could be applied to a wide variety of candidate genes as it leads to therapeutic effect reiterations and increases safety by allowing careful therapeutic adjustments.

Pulmonary arterial hypertension (PAH) is one of the leading causes of morbidity and mortality in adult patients with sickle cell disease (SCD). Here, we developed a model to study the early stage of PAH in SCD. We exposed wild-type and transgenic sickle cell SAD (Hbb(s)/Hbb(s)) mice to hypoxia (8% O(2)) for 7 days. Prolonged hypoxia in SAD mice only induced 1) increased neutrophil count in both bronchoalveolar lavage (BAL) and peripheral circulation; 2) increased BAL IL1beta, IL10, IL6, and TNF-alpha; and 3) up-regulation of the genes endothelin-1, cyclo-oxygenase-2, angiotensin-converting-enzyme, and IL-1beta, suggesting that amplified inflammatory response and activation of the endothelin-1 system may contribute to the early phase of PAH in SCD. Since phosphodiesterases (PDEs) are involved in pulmonary vascular tone regulation, we evaluated gene expression of phosphodiesterase-4 (PDE-4) isoforms and of PDE-1, -2, -3, -7, -8, which are the main cyclic-adenosine-monophosphate hydrolyzing enzymes. In SAD mouse lungs, prolonged hypoxia significantly increased PDE-4 and -1 gene expressions. The PDE-4 inhibitor, rolipram, prevented the hypoxia-induced PDE-4 and -1 gene up-regulation and interfered with the development of PAH, most likely through modulation of both vascular tone and inflammatory factors. This finding supports a possible therapeutic use of PDEs inhibitors in the earlier phases of PAH in SCD.

Background: In patients with hemoglobinopathies, hematopoietic stem cell (HSC) gene therapy has the potential to induce production of functional β-globin in the red blood cell lineage with the aim of reducing or eliminating the symptoms of disease. Previous results from 1 subject with severe sickle cell disease (SCD; 6 months follow-up) and 2 subjects with β0/βE-thalassemia major (up to 15 months follow-up) treated in clinical study HGB-205 suggested that transplantation with autologous CD34+ cells transduced with the LentiGlobin BB305 lentiviral vector containing an engineered βA-T87Q-globin gene (LentiGlobin BB305 Drug Product) resulted in near-normal levels of total hemoglobin (Hb) and rapid clinical improvement. Here we provide data on a new subject enrolled and additional follow-up data on the 3 subjects previously presented in Study HGB-205. Subjects and Methods: Subjects with severe SCD underwent HSC collection via bone marrow harvest, while subjects with β-thalassemia major underwent HSC collection via peripheral blood apheresis following mobilization. CD34+ cells were selected and transduced with LentiGlobin BB305 lentiviral vector to produce the drug product. Subjects underwent myeloablation with intravenous busulfan, followed by infusion of drug product. Subjects were monitored for hematological engraftment, vector copy number, βA-T87Q-globin expression, adverse events and transfusion requirements. Integration site analysis (ISA) and replication-competent lentivirus (RCL) assays were performed. Prophylactic RBC transfusions were continued in subjects with SCD who were on chronic transfusion pre-transplant to maintain HbS Results: As of 31 July 2015, 1 subject with severe SCD (Subject 1204, βS/βS with multiple vaso-occlusive crises, silent infarct, acute chest syndrome, and on prophylactic transfusions) and 3 subjects withβ-thalassemia major (Subjects 1201, 1202 and 1203) have been infused with the LentiGlobin BB305 Drug Product. The outcome of these subjects to date is shown in Table 1. No subject has experienced a drug product-related adverse event, and ISA analyses demonstrate highly polyclonal reconstitution without clonal dominance. The subject with severe SCD is producing approximately 51.5% of anti-sickling hemoglobin (48% HbAT87Q, 1.8% HbF, 1.7% HbA2) at 9 months post-infusion. This subject has not had a post-infusion hospitalization for a SCD-related event despite stopping chronic transfusions at Day +88. Both subjects with β0/βE-thalassemia major have remained transfusion-free for at least 15 months post-infusion, with a consistent expression of βA-T87Q-globin; the subject with β0/β0-thalassemia major has only had 1 month follow-up post-drug product infusion to date. Conclusion: The subject with severe SCD is producing approximately 51.5% anti-sickling globins with HbS of 48.5% and remains free of SCD-related events despite stopping chronic transfusion therapy. Two subjects with β0/βE-thalassemia major remain transfusion-free for at least 15 months post infusion of LentiGlobin BB305 Drug Product. Gene therapy using autologous HSC transduced with LentiGlobin BB305 lentiviral vector is a promising approach for the treatment of patients with hemoglobinopathies. Table 1. Demographics and Transplantation Outcomes Subject Age (years)/ Sex (M/F) Genotype BB305 Drug Product Day of Neutrophil Engraftment Drug Product- related Adverse Events Day of Last pRBC Transfusion Last Study Visit (Months) Hb at Last Visit (g/dL) VCNa CD34+ cell dose (x106 per kg) Subject with severe sickle cell disease HbAT87Q/HbF/ HbS/Total Hb 1204 13/ M βS/βS 1.2 / 1.0 5.6 Day +37 None Day +88 9M 5.5/0.2/5.5/11.4 Subjects with β-thalassemia major Hb AT87Q/ Total Hb 1201 18/ F β0/βE 1.5 8.9 Day +13 None Day +10 18M 7.8/10.7 1202 16/ M β0/βE 2.1 13.6 Day +15 None Day +12 15M 9.7/12.8 1203 19/ M β0/β0 0.8 8.8 Day +28 None Day +15 1M Pending/9.2 As of 31 July 2015 aVCN, vector copy number; F=female; M= Male for gender, and months for day of last follow-up Disclosures Payen: bluebrid bio: Consultancy. Beuzard:bluebird bio Inc: Consultancy, Equity Ownership. von Kalle:bluebird bio, Inc.: Consultancy. Sandler:bluebird bio, Inc.: Employment, Equity Ownership. Soni:bluebird bio, Inc.: Employment, Equity Ownership. De Montalembert:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Leboulch:bluebird bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Background : In patients with β-thalassemia major, hematopoietic stem cell (HSC) gene therapy has the potential to induce production of β-globin, γ-globin or modified β-globin in the red blood cell lineage and reduce or stop the need for blood transfusions. We have previously presented early results for 2 subjects with β/βE -thalassemia major that suggested that transplantation with autologous CD34+ cells transduced with a replication-defective, self-inactivating LentiGlobin BB305 lentiviral vector containing an engineered β-globin gene (βA-T87Q) resulted in near-normal levels of total hemoglobin (Hb) early after HSC infusion. Herein, we provide additional follow-up data on these two subjects. Subjects and Methods : After obtaining 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 quantitative PCR performed on pooled colony-forming progenitors. Subjects underwent myeloablation with intravenous busulfan, followed by infusion of transduced CD34+ cells. Subjects were monitored for hematological engraftment, βA-T87Q-globin expression (by high performance liquid chromatography) and transfusion requirements. Integration site analysis (ISA, by linear amplification-mediated PCR and high-throughput sequencing on nucleated cells) and replication-competent lentivirus (RCL) assays were performed. Results: As of 31 July 2014, two subjects with β0/βE thalassemia major (Subjects 1201 and 1202) have undergone infusion with drug product. The outcome of these two subjects to date is shown in Table 1. The initial safety profile is consistent with myeloablation, without serious adverse events or drug product-related adverse events. Both subjects remain transfusion independent. ISA analyses in both the subjects at 3 months shows polyclonal reconstitution. An additional 2 subjects have been enrolled in this study but have not yet undergone drug product infusion. Conclusion: In the first two subjects, early transfusion independence was achieved and has been maintained as of 31 July 2014. Further follow up data on these two subjects and additional data on subjects who have undergone drug product infusion in this study will be presented. Gene therapy using autologous HSC transduced with LentiGlobin BB305 lentiviral vector is a promising approach for the treatment of patients with β-thalassemia major. | Subject | Age (years) and gender | Genotype | BB305 Drug Product | Day of Neutrophil Engraftment | Drug Product-related Adverse Events | Day of last pRBC transfusion | Day of last follow up | βA-T87Q-Hb at last follow-up visit /Total Hb (g/dL) | | ------- | ----------------------------- | -------- | ------------------ | ----------------------------- | ----------------------------------- | ---------------------------- | --------------------- | --------------------------------------------------- | -------- | | VCNa | CD34+ cell dose (x106 per kg) | | 1201 | 19 F | β/βE | 1.5 | 8.9 | Day +13 | None | Day +10 | Day +180 | 7.2/10.2 | | 1202 | 16 M | β/βE | 2.1 | 13.6 | Day +15 | None | Day +12 | Day +90 | 6.8/11.0 | Abstract 4797. Table 1. Preliminary Results of Dosing Parameters and Transplantation Outcomes As of 31 July 2014 a VCN, mean vector copy number Disclosures Payen: bluebird bio, Inc: Consultancy. Beuzard: bluebird bio, Inc: Consultancy, Equity Ownership. Sandler: bluebird bio, Inc: Employment, Equity Ownership. Soni: bluebird bio, Inc.: Employment, Equity Ownership. De Montalembert: Novartis : Speakers Bureau. Leboulch: bluebird bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Murine ZFP-37 is a member of the large family of C2H2 type zinc finger proteins. It is characterized by a truncated NH2-terminal Krüppel-associated box and is thought to play a role in transcriptional regulation. During development Zfp-37 mRNA is most abundant in the developing central nervous system, and in the adult mouse expression is restricted largely to testis and brain. Here we show that at the protein level ZFP-37 is detected readily in neurons of the adult central nervous system but hardly in testis. In brain ZFP-37 is associated with nucleoli and appears to contact heterochromatin. Mouse and human ZFP-37 have a basic histone H1-like linker domain, located between KRAB and zinc finger regions, which binds double-stranded DNA. Thus we suggest that ZFP-37 is a structural protein of the neuronal nucleus which plays a role in the maintenance of specialized chromatin domains.

In mammals, the presence of SRY, the sex-determining gene located on the Y chromosome is required to induce the gonadal anlage to differentiate as a testis, whereas its absence leads to the development of an ovary. We report here the characterization by 5' and 3' RACE analysis of several SRY transcripts which are expressed in the ovine male developing gonads. These transcripts were not detected in any other fetal tissues and were expressed only in the genital portion of the urogenital ridge. The temporal profile of SRY expression analyzed by RT-PCR suggests that in the sheep fetus the role of SRY is not limited to initiating Sertoli cell differentiation as in mice. Indeed, SRY transcripts persist after the full differentiation of the testis. In addition to SRY, other genes are known to be involved in mammalian sex determination: Wilms' tumor gene WT-1, steroidogenic factor gene Ftz-F1 (SF-1) and anti-Müllerian hormone (AMH). We investigated the expression patterns of these genes by RT-PCR during fetal development in sheep gonads. Concerning WT-1 and SF-1, our results are consistent with those described in mice where the earliest expression was detected before the sexual differentiation in both sexes. In male, the ontogenesis of AMH transcription corresponds to the seminiferous cords formation (30 dpc). In female, we have observed the presence of SF-1 transcripts from the undifferentiated stage until birth. In addition, P450 aromatase expression is detected from 30 dpc and is correlated with the presence of 17-beta estradiol in sheep ovary. These data reveal significant differences between rodent and ruminant models concerning the sex-determining pathway.

A microsatellite in the WT (Wilms tumour) gene is shown to be evolutionarily conserved in a range of mammals. The microsatellite was monomorphic in pig and two alleles have been found in goat. In cattle, a one-base size polymorphism has been discovered outside the microsatellite. WT was genetically mapped to bovine chromosome 15 and to sheep chromosome 15 by synteny mapping. Conservation of chromosome segments of HSA11 and BTA15 is discussed.

Abstract 247 Background: β-thalassemia is associated with ineffective erythropoiesis, accelerated erythroid differentiation and apoptosis resulting in anemia and iron overload. The molecular mechanism involved is still incompletely understood. Members of the TGF-β superfamily participate in both proliferation and differentiation of erythroid progenitors. However, the role of these molecules in models of ineffective erythropoiesis has not been addressed so far. RAP-011 is a ligand trap consisting of the extracellular domain of ActRIIA linked to mouse IgG1 Fc domain. We aimed to study the role of ActRIIA signaling in the ineffective erythropoiesis of β-thalassemia and to evaluate the therapeutic impact of RAP-011. Methods: Hbbth1/th1 mice (a model of β-thalassemia intermedia) were subcutaneously treated with RAP-011 (10mg/kg body weight) twice a week for 30–60 days and biological and biochemical parameters were followed. Results: RAP-011 treatment significantly increased hemoglobin levels, red blood cell counts, MCV, MCH and hematocrit with a concomitant decrease in bilirubin levels and reticulocyte counts (since 10 days of treatment and sustained until day 60 of follow up). Flow cytometry analysis showed that RAP-011 significantly decreased late basophilic and polychromatic erythroblast cell numbers in both bone marrow and spleen indicating that RAP-011 corrects ineffective erythropoiesis. We next evaluated the expression of putative ActRIIA ligand(s) in β-thalassemia. Increased expression of Growth Differentiation Factor 11 (GDF11) was observed in cultured erythroblasts and in spleen sections of thalassemic mice. RAP-011 treatment decreased these elevated GDF11 levels in both bone marrow and spleen. We further investigated how BMP/Activin signaling was involved in ineffective erythropoiesis. Anti-GDF11 antibodies, follistatin (activin and GDF11 antagonist) and dorsomorphin (a small molecule inhibitor of SMAD1/5/8 phosphorylation) reduced differentiation, induced FAS-L expression and apoptosis in erythroblasts both in vivo and in vitro whereas noggin (a BMP-2/4 antagonist) had no effect on erythroblast differentiation. Altogether, these data suggest that Activin/BMP signaling controls erythroblast differentiation and targeting BMP type II /activin type II receptors can decrease ineffective erythropoiesis of β-thalassemia. Summary: Sotatercept (a humanized version of RAP-011) is currently in phase II clinical trials for treatment of anemia in patients with Myeloma Bone Disease and End Stage Renal Disease and data from our non-clinical findings support a newly initiated β-thalassemia clinical trial. Our results suggest that sotatercept would be a potential therapeutic tool to improve anemia, increase hemoglobin levels and correct ineffective erythropoiesis and its side effects in β-thalassemic patients. Disclosures: Daniel: Celgene Corporation: Employment. Chopra:Celgene Corp: Employment, Equity Ownership. Sung:Celgene: Employment.