Increased fetal hemoglobin (HbF) levels diminish the clinical severity of ␤-thalassemia and sickle cell anemia. A treatment strategy using autologous stem celltargeted gene transfer of a ␥-globin gene may therefore have therapeutic potential. We evaluated oncoretroviral-and lentiviral-based ␥-globin vectors for expression in transduced erythroid cell lines. Compared with ␥-globin, oncoretroviral vectors containing either a ␤-spectrin or ␤-globin promoter and the ␣-globin HS40 element, a ␥-globin lentiviral vector utilizing the ␤-globin promoter and elements from the ␤-globin locus control region demonstrated a higher probability of expression. This lentiviral vector design was evaluated in lethally irradiated mice that received transplants of transduced bone marrow cells. Long-term, stable erythroid expression of human ␥-globin was observed with levels of vector-encoded ␥-globin mRNA ranging from 9% to 19% of total murine ␣-globin mRNA. The therapeutic efficacy of the vector was subsequently evaluated in a murine model of ␤-thalassemia intermedia. The majority of mice that underwent transplantation expressed significant levels of chimeric m␣ 2 h␥ 2 molecules (termed HbF), the amount of which correlated with the degree of phenotypic improvement. A group of animals with a mean HbF level of 21% displayed a 2.5 g/dL (25 g/L) improvement in Hb concentration and normalization of erythrocyte morphology relative to control animals. ␥-Globin expression and phenotypic improvement was variably lower in other animals due to differences in vector copy number and chromosomal position effects. These data establish the potential of using a ␥-globin lentiviral vector for gene therapy of ␤-thalassemia. IntroductionThe hemoglobin disorders are highly prevalent, recessive genetic diseases in which coinheritance of 2 defective globin alleles results in severe hematologic disease. In patients with sickle cell anemia, the beta chain of hemoglobin S contains a substitution of valine for glutamic acid at position 6. 1 This substitution results in a change in surface charge that predisposes deoxygenated HbS to polymerize, causing red cells to assume rigid sickled shapes leading to vaso-occlusion, painful crisis, and organ damage. Defective synthesis of ␤-globin in patients with severe ␤-thalassemia due to a variety of mutational mechanisms leads to the accumulation of aggregates of unpaired, insoluble ␣-chains that cause ineffective erythropoiesis, accelerated red cell destruction, and severe anemia. 2 Although palliative therapies improve the quality and duration of life for many individuals, overall treatment for these disorders remains unsatisfactory. A few patients with sickle cell disease and a somewhat larger number with ␤-thalassemia have been cured with bone marrow (BM) transplantation from HLAmatched siblings, but such treatment is available for only a small minority of patients. 3,4 These considerations have made the development of gene therapy for hemoglobin disorders a highly desired goal.Effective gene therapy for hemoglobi...

Substantial effort has been invested in developing methodologies for efficient gene transfer into human, repopulating, hematopoietic stem cells. Oncoretroviral vectors are limited by the lack of nuclear mitosis in quiescent stem cells during ex vivo transduction, whereas the preintegration complex of lentiviral vectors contains nuclear-localizing signals that permit genome integration without mitosis. We have developed a flexible and versatile system for generating lentiviral vector particles and have pseudotyped such particles with amphotropic, ecotropic, feline endogenous virus (RD114) or vesicular stomatitis virus (VSV-G) envelope proteins. Particles of all four types could be concentrated approximately 100-fold by ultracentrifugation or ultrafiltration. RD114 or amphotropic particles were more efficient than VSV-G-pseudotyped particles at transducing human cord blood CD34(+) cells and clonogenic progenitors within that population. Amphotropic particles transduced cytokine-mobilized, human peripheral blood CD34(+) cells capable of establishing hematopoiesis in immunodeficient mice more efficiently than the other two types of particles. We conclude that the use of amphotropic pseudotyped lentiviral vector particles rather than the commonly used VSV-G-pseudotyped particles should be considered in potential applications of lentiviral vectors for gene transfer into this therapeutically relevant target cell population.

Since increased fetal hemoglobin diminishes the severity of ␤-thalassemia and sickle cell anemia, a strategy using autologous, stem cell-targeted gene transfer of a ␥-globin gene may be therapeutically useful. We previously found that a ␥-globin lentiviral vector utilizing the ␤-globin promoter and elements from the ␤-globin locus control region (LCR) totaling 1.7 kb could correct murine ␤-thalassemia. However, therapeutic consistency was compromised by chromosomal position effects on vector expression. In contrast, we show here that the majority of animals that received transplants of ␤-thalassemic stem cells transduced with a new vector containing 3.2 kb of LCR sequences expressed high levels of fetal hemoglobin (17%-33%), with an average vector copy number of 1.3. This led to a mean 26 g/L (2.6 g/dL) increase in hemoglobin concentration and enhanced amelioration of other hematologic parameters. Analysis of clonal erythroid cells of secondary spleen colonies from mice that underwent transplantation demonstrated an increased resistance of the larger LCR vector to stable and variegating position effects. This trend was also observed for vector insertion sites located inside genes, where vector expression was often compromised, in contrast to intergenic sites, where higher levels of expression were observed. These data emphasize the importance of overcoming detrimental position effects for consistent therapeutic globin vector expression.

Increased levels of red cell fetal hemogloblin, whether due to hereditary persistence of expression or from induction with hydroxyurea therapy, effectively ameliorate sickle cell disease. Therefore, we developed erythroid-specific, γ-globin lentiviral vectors for hematopoietic stem cell-targeted gene therapy with the goal of permanently increasing HbF production in sickle red cells. We evaluated two different γ-globin lentiviral vectors for therapeutic efficacy in the BERK sickle cell mouse model. The first vector, V5, contained the γ-globin gene driven by 3.1 kb of β-globin regulatory sequences and a 130 bp β-globin promoter. The second vector, V5m3, was identical except that the γ-globin 3'UTR was replaced with the β-globin 3'UTR. Adult erythroid cells have β-globin mRNA 3’UTR binding proteins that enhance β-globin mRNA stability and we postulated this design might enhance γ-globin expression. Stem cell gene transfer was efficient and nearly all red cells in transplanted mice expressed human γ-globin. Both vectors demonstrated efficacy in disease correction, with the V5m3 vector producing a higher level of γ-globin mRNA which was associated with high level correction of anemia and secondary organ pathology. These data support the rationale for a gene therapy approach to sickle cell disease by permanently enhancing HbF using a γ-globin lentiviral vector.

β-Thalassemia major results from severely reduced or absent expression of the β-chain of adult hemoglobin (α2β2;HbA). Increased levels of fetal hemoglobin (α2γ2;HbF), such as occurs with hereditary persistence of HbF, ameliorate the severity of β-thalassemia, raising the potential for genetic therapy directed at enhancing HbF. We used an in vitro model of human erythropoiesis to assay for enhanced production of HbF after gene delivery into CD34+ cells obtained from mobilized peripheral blood of normal adults or steady-state bone marrow from patients with β-thalassemia major. Lentiviral vectors encoding (1) a human γ-globin gene with or without an insulator, (2) a synthetic zinc-finger transcription factor designed to interact with the γ-globin gene promoters, or (3) a short-hairpin RNA targeting the γ-globin gene repressor, BCL11A, were tested. Erythroid progeny of normal CD34+ cells demonstrated levels of HbF up to 21% per vector copy. For β-thalassemic CD34+ cells, similar gene transfer efficiencies achieved HbF production ranging from 45% to 60%, resulting in up to a 3-fold increase in the total cellular Hb content. These observations suggest that both lentiviral-mediated γ-globin gene addition and genetic reactivation of endogenous γ-globin genes have potential to provide therapeutic HbF levels to patients with β-globin deficiency.