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BACKGROUND: Donor availability and transplantation-related risks limit the broad use of allogeneic hematopoietic-cell transplantation in patients with transfusion-dependent ß-thalassemia. After previously establishing that lentiviral transfer of a marked ß-globin (ß(A-T87Q)) gene could substitute for long-term red-cell transfusions in a patient with ß-thalassemia, we wanted to evaluate the safety and efficacy of such gene therapy in patients with transfusion-dependent ß-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 ß-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-ß(0)/ß(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 ß(0)/ß(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 ß-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 .).

K562 cell line was grown in liquid suspension and in plasma clot cultures. Morphological studies revealed the presence of a minority of cells, which were identified as erythroblasts. However, the majority of the cells remained unidentified. Biochemical studies confirmed the synthesis of hemoglobin by K562 cells. The pattern of hemoglobin (Hb) production was of the embryonic type, with the presence of small amount of fetal Hb. The addition of several inducers, like Epo and butyrate, was unable to modify the pattern of Hb production of K562. In contrast, the addition of hemin increased the synthesis of Hb and stimulated the synthesis of fetal Hb and probably adult Hb.

The G gamma and A gamma content of Hb F produced in cultures of BFU-Es from the blood of normal fetuses, neonates, and adults was determined. The results show that erythroid progenitors produce A gamma and G gamma chains in a ratio characteristic of their ontogenic stage. The analysis of the G gamma/A gamma ratio in culture of BRU-Es from thalassemic patients showed a marked heterogeneity, resembling that observed in freshly drawn cells. These results afford evidence that the type of gamma chain produced is programmed at the level of early erythroid progenitors.

Repeated injections of large doses of erythropoietin (Epo) have been shown to be of benefit in the treatment of murine and human beta-thalassemia. To determine whether Epo gene therapy could replace this treatment for long-term periods, lethally irradiated beta-thalassemic (Hbbd3th haplotype) and normal DBA/2J (Hbbd haplotype) mice were grafted with syngeneic bone marrow cells infected with a retroviral vector carrying the Epo cDNA. In normal mice, dysregulated Epo production induced elevated serum Epo levels (176 +/- 68 mU/mL), high hematocrit levels (73% +/- 8%), and elevated beta-minor globin chain synthesis. In contrast, in thalassemic mice, moderate increases in the hematocrit levels (from 33% +/- 1% to 43% +/- 9%), associated with limited increases in the initially elevated Epo levels (from 83 +/- 22 to 190 +/- 230 mU/mL), were recorded 2 months after transplantation. In mice in which the hematocrit increased most, from 33% +/- 1% before transplantation to 49% +/- 10%, the retroviral Epo gene expression induced a striking improvement of the beta-thalassemic syndrome. These mice exhibited normal or near-normal beta/alpha-globin chain synthesis ratios, induced by the activation of the beta-minor chain. This led to the elimination of the high amounts of unpaired alpha chains in erythrocytes and finally reduced the reticulocyte count despite the permanent Epo stimulation. These results show that efficient Epo gene expression corrects the erythrocyte phenotype of the mouse beta-thalassemic syndrome. However, the incidence of lethal polycythemia or of transient improvements indicates that the present strategy is only the first step toward such indirect gene therapy.

Organoids are adenoviral vector transduced cells embedded ex vivo in a collagen-polytetrafluoroethylene lattice that is saturated with angiogenic factors. Organoids provide an alternative method of cell mediated gene transfer following implantation in the donor/recipient. The feasibility of adenovirally mediated delivery via organoids using the erythropoietin (Epo) cDNA was tested. Fibroblasts were transduced by two recombinant adenoviral vectors encoding the Macaca cynomolgus Epo cDNA, driven by a viral (RSV LTR) or a murine housekeeping gene promoter (PGK-1). A functional in vivo assay was used to monitor Epo production via the rise in hematocrit(s) (hct). The hct remained elevated for as long as 6 weeks after implantation. Subcutaneous implants gave consistently higher hct than intraperitoneal implants, while organoids made with a greater number of cells, or an equal number of cells transduced at higher multiplicities of infection (MOI) also produced a larger increase in hct. AdPGKEpo-organoids produced a greater increase in hct than AdRSVEpo-organoids under comparable conditions, but the duration of expression was similar. A 10- to 50-fold lower input of AdRSVEpo using organoids versus direct intravenous injections resulted in an equal to, or greater than hct response in mice. Explanted organoids caused a rapid decrease in the hct of mice. Organoid supernatant had little or no detectable free viral particles making this method safe from unwanted recombinant adenovirus dissemination.

The perturbations of the conformation of human deoxyhemoglobin induced by the covalent attachment of glutathione at cysteine beta 93 have been investigated by computer simulation in conjunction with molecular graphics. In the first phase of the analysis, a systematic search was carried out of the conformational space of glutathione attached to deoxyhemoglobin. In this search, the conformation of the hemoglobin molecule was held constant, while the relative energies of a series of 186,624 glutathione conformations involving systematic variation of six dihedral angels were calculated. From this search, the most favorable conformation was selected as the starting conformation for energy minimization of the glutathionyl hemoglobin molecule as a function of all Cartesian coordinates. In order to provide a reference state, an independent minimization by the same procedures was carried out for deoxyhemoglobin in the absence of glutathione. Comparison of the minimized structures with and without glutathione attached revealed a number of significant differences. The most conspicuous difference in the protein moiety concerned the salt bridge between aspartate beta 94 and histidine beta 146 which is destabilized upon minimization of the glutathionyl-hemoglobin complex due to interactions of the aspartate residue with the glycyl NH group of glutathione. Other observed differences in the minimized structures are located at the alpha 1-beta 2 interface and include displacement of the carboxyl group of aspartate beta 99. In the minimized complex, the glutathione portion assumes a quasi-cyclic conformation stabilized through interactions between the free (gamma-glutamyl) amino and (glycyl) carboxyl ends of the tripeptide and between this carboxyl end and the epsilon amino group of lysine alpha 40. In a parallel conformational study of glutathione alone, a similar structure was found as the lowest energy form. These quasi-cyclic conformations contrast with the extended structures reported by Wright (Wright, W.B. (1955) Acta Crystallogr. 11, 632-642) for crystals of glutathione where interactions between molecules play a major role. The conclusions of our analysis are in agreement with the experimental investigations reported in the two preceding papers and permit, moreover, a coherent interpretation of the observed functional and structural changes in deoxyhemoglobin induced by glutathione.