Sickle cell disease (SCD) is caused by a single point mutation in the human betaA globin gene that results in the formation of an abnormal hemoglobin [HbS (alpha2betaS2)]. We designed a betaA 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.

We have mapped the distribution of the major and minor DNase I-hypersensitive sites in the human "fi-like-globin" gene domain. The minor DNase I-hypersensitive sites map close to the 5' end of each of the 13-like-globin genes. Their presence is specifically associated with the transcription of the immediate downstream fi-like-globin genes.The major DNase I-hypersensitive sites map in what appear to be the 5' and 3' boundary areas of the human fi-like-globin gene domain, a region estimated to span at least 90 kilobases of DNA. These major sites are present in various erythroid cells, which express predominantly either the embryonic, the fetal, or the adult (3-like-globin genes, and seem to be involved in derming the active j8-like-globin gene domain in cells of erythroid lineage. The four major DNase I-hypersensitive sites in the 5' boundary area, when correlated with sequencing data, are shown to be located in DNA regions containing enhancer core-like sequences and alternating purine and pyrimidine bases.The human "/3-like-globin" genes (hemoglobin P-chain gene cluster) encode, respectively, one embryonic (e), two fetal (G y and Ay), and two adult (8 and /3) globin chains. These genes have been shown to reside within "50 kilobases (kb) of chromosomal DNA in the transcriptional order 5' e-y.GAy. EXPERIMENTAL PROCEDURES Cells were grown as described (7). Human bone marrow cells were collected from cancer patients with normal marrow who were to undergo chemotherapy and bone marrow reinfusion. Isolated by dextran column chromatography, -25% of the nucleated cells were erythroid.DNase I-digestion, gel electrophoresis, RNA isolation, blotting, and hybridization were carried out as described (7). RESULTS Globin Gene Transcription in K562,HEL, Adult Human Marrow, and HL60 Cells. Nuclear and cytoplasmic RNAs were isolated from cells, and individual globin gene transcription was detected by "dot-blot" hybridization with e-,

No abstract