Treatment with Azacitidine of Patients with End-Stage β-Thalassemia

Lowrey, Christopher H.; Nienhuis, Arthur W.

doi:10.1056/nejm199309163291205

Cited by 119 publications

(68 citation statements)

References 25 publications

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“…The only direct side effect of aza-C was a dose-dependent neutropenia, which was compensated by reducing the size or frequency of the aza-C dose. As aza-C is well tolerated in vivo under experimental conditions (as our results show) and in humans, 15 it might be a valid way of overcoming methylation and promoting stable gene expression and long-term benefits.…”

Section: Figure 2 Tumour Onset In Scid Mice Tumours Were Induced In supporting

confidence: 68%

“…Previous clinical use of aza-C, 15 administered at 2 mg/kg body weight per day by continuous intravenous infusion for 4 days at 2-to 4-week intervals, has produced long-term therapeutic benefits in patients with end-stage ␤-thalassemia. The only direct side effect of aza-C was a dose-dependent neutropenia, which was compensated by reducing the size or frequency of the aza-C dose.…”

Section: Figure 2 Tumour Onset In Scid Mice Tumours Were Induced In mentioning

confidence: 99%

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5-Azacytidine prevents transgene methylation in vivo

et al. 1999

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Retroviral sequence can silence transgene expression in C, maintained the high expression of lacZ and tk genes at vitro and in vivo. We report that this effect can be efficiently the baseline values. LacZ-positive cells in the tumour prevented by in vivo administration of the demethylating masses after death was weak (1-2%) in the control group, agent 5-azacytidine (aza-C). We engineered the U937 while in mice treated with aza-C it was maintained at 90%. human cell line with a retroviral vector consisting of the The delay in tumour onset was significanly longer when thymidine kinase suicide gene (tk), which induces sensianimals were treated with both aza-C and gcv tivity to ganciclovir (gcv) and through an IRES sequence, (P Ͻ 0.0001) compared with animals treated with gcv or the bacterial beta-galactosidase gene (lacZ) as a marker with aza-C alone. The prevention of silencing phenomena gene. About 90% of the U937 cells expressed the transhas important implications for gene therapy, because an gene. By injecting the transduced U937 cells in severe efficient transduction associated with appropriate drug combined immunodeficient disease (SCID) mice, we gentherapy, might be a powerful strategy for successful erated a tumour which, during in vivo treatment with azaapplication of gene therapy protocols.Keywords: 5-azacytidine; DNA methylation; SCID mice; retroviral vectors Retroviral vectors efficiently insert exogenous genes into a wide range of human cells and have been extensively studied in an attempt to develop gene therapy protocols. 1,2 Although gene transfer is generally successful, high level, long-term expression in primary cells is problematic. One of the major drawbacks is in vitro and in vivo inhibition of transgene expression because of DNA methylation. 3,4 The DNA demethylating agent 5′ azacytidine (aza-C) is reported to reactivate transgene expression. 5,6 We report inhibition of transgene expression occurs in the stably infected U937 human cell line in vitro and in vivo and aza-C treatment prevents DNA methylation. We used a bicistronic retroviral vector to transduce the U937 human cell line. Through an internal ribosome entry site (IRES) 7 sequence the vector cotransfers the herpes simplex virus type 1 thymidine kinase gene (HSVtk), which induces sensitivity to ganciclovir (gcv), and a marker gene, the bacterial beta-galactosidase gene (lacZ) (Figure 1a). By injecting the HSVtk/lacZ transduced U937 cells in mice with severe combined immunodeficient disease (SCID), we generated a tumor which, when the animal was killed, was almost completely methylated. In vivo treatment with aza-C prevented transgene methylation, as demonstrated by increased lacZ expression and gcv sensitivity. When aza-C was combined with gcv, tumor onset was significantly delayed. Our study shows aza-C prevents DNA methylation. This may have implications for gene therapy in genetic and neoplastic diseases.As previously reported, 8 by transferring MFG/tk-IRESlacZ into the GP+envAM12 cell line, we obtained an amphotropic producing ce...

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Section: Figure 2 Tumour Onset In Scid Mice Tumours Were Induced In supporting

confidence: 68%

Section: Figure 2 Tumour Onset In Scid Mice Tumours Were Induced In mentioning

confidence: 99%

5-Azacytidine prevents transgene methylation in vivo

et al. 1999

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“…However, because the half-life of this agent is only a few hours, we administered it on a daily basis in a schedule which is similar to those used to treat patients with leukemia, myelodysplastic syndromes and the hemoglobinopathies. [37][38][39]43,44 In some reports 5-Aza concentrations as high as 4-5 m have been used to increase gene expression. 2,31,33 Yet, at these concentrations we noted markedly decreased cell viability and no reactivation of expression.…”

Section: Discussionmentioning

confidence: 99%

“…Because 5-Aza has a short half-life it is typically administered to patients by continuous infusion or daily subcutaneous injection over several days. [37][38][39]43,44 To model these treatment schedules we treated our cell cultures on a daily basis. Similarly, because our goal was to study potentially clinically relevant effects, we initially chose 5-Aza concentrations of 250 nm for the treatment of MEL cells and Representative results of the ␤-gal staining assay are shown in Figure 5.…”

Section: -Azacytidine and Tsa Treatment Of Silenced Clonesmentioning

confidence: 99%

“…36 This agent is used clinically to treat leukemia and the myelodysplastic syndromes and has been used experimentally to treat ␤-thalassemia and sickle cell disease. [37][38][39] Some of the earliest applications of 5-Aza to the study of retroviral silencing demonstrated that 5-Aza was able to revert the MMLV provirus to an infectious state after its expression had been silenced in EC cells and mice. 6,30 While 5-Aza has since been successfully used to reactivate silenced gene expression from MMLV-based retroviral vectors in a variety of cell lines and experimental contexts, most experiments have focused on short-term silencing which takes place within days following retroviral transduction.…”

Section: Introductionmentioning

confidence: 99%

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Long-term silencing of retroviral vectors is resistant to reversal by trichostatin A and 5-azacytidine

2000

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One problem limiting the development of long-term gene replacement therapy is gene silencing. A variety of experiments have implicated DNA methylation and histone deacetylation in gene silencing and shown that the agents 5-azacytidine (5-Aza) and trichostatin A (TSA) are able to reverse these effects. To begin to investigate clinically relevant strategies to reverse silencing with these drugs, we transduced the MEL and FDCP-1 hematopoietic cell lines with Moloney murine leukemia virus (MMLV) and Harvey murine sarcoma virus (HMSV)-based retroviral vectors carrying the ␤-galactosidase/neomycin resistance fusion gene (␤-geo). Fifty-one clones were isolated under G418 selection over 2 weeks and then allowed to grow without selection as

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In vitro effect on human leukemic K562 cells of co-administration of liposome-associated retinoids and cytosine arabinoside (ara-C)

et al. 1999

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The administration of retinoids has been demonstrated to be of potential utility in the therapy of a wide spectrum of neoplastic pathologies due to the ability to induce differentiation in a large variety of primary tumor cells as well as in vitro cultured cell lines. Moreover, a number of compounds, including hemin, cytosine arabinoside, and 5-azacytidine are able to induce erythroid differentiation of the erythroleukemic cell line K562. In this paper we determined whether a combined treatment of K562 cells with suboptimal concentrations of cytosine arabinoside and retinoids containing liposomes lead to a full expression of differentiated functions. Liposomes were prepared by reverse phase evaporation technique followed by extrusion through polycarbonate filters. Cell growth kinetics studies and intracellular detection of hemoglobin by benzidine staining were performed. The results obtained showed that the combined treatment with liposomes containing retinoids and sub-optimal concentration of ara-C is an effective strategy to induce K562 cell differentiation, minimizing at the same time toxic effects. Control experiments aimed to determine possible selection of subpopulations of K562 cells suggest that the observed results are not related to toxicity and/or potential selection of induced cells. In conclusion, liposomally delivered retinoids could be proposed for differentiation therapy as an effective strategy in the treatment and management of malignancy. In addition, the finding that liposomally delivered retinoids increase the capacity of cytosine arabinoside to induce erythroid differentiation, could be of interest in studies aimed at the development of treatment able to reactivate fetal globin genes in beta-thalassemia patients.

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Treatment with Azacitidine of Patients with End-Stage β-Thalassemia

Cited by 119 publications

References 25 publications

5-Azacytidine prevents transgene methylation in vivo

5-Azacytidine prevents transgene methylation in vivo

Long-term silencing of retroviral vectors is resistant to reversal by trichostatin A and 5-azacytidine

In vitro effect on human leukemic K562 cells of co-administration of liposome-associated retinoids and cytosine arabinoside (ara-C)

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