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...

Nitrosoureas form O6-alkylguanine-DNA adducts that are converted to G to A transitions, the mutation found in the activated ras oncogenes of nitrosourea-induced mouse lymphomas and rat mammary tumors. These adducts are removed by the DNA repair protein O6-alkylguanine-DNA alkyltransferase. Transgenic mice that express the human homolog of this protein in the thymus were found to be protected from developing thymic lymphomas after exposure to N-methyl-N-nitrosourea. Thus, transgenic expression of a single human DNA repair gene is sufficient to block chemical carcinogenesis. The transduction of DNA repair genes in vivo may unravel mechanisms of carcinogenesis and provide therapeutic protection from known carcinogens.

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