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Query: UMLS:C0002895 (sickle cell disease)
 11,747 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Gene therapy for the beta thalassemias and sickle cell anemia will require high levels of expression of human beta globin genes. One method to achieve this goal is amplification of globin genes transferred into the stem cells in the bone marrow of these patients. If the amplified genes remain normally regulated, they will then further increase their expression on being induced to differentiate along an erythroid pathway. To begin this study, we constructed a plasmid containing a neomycin resistance gene, a human beta globin gene, and a wild-type DHFR cDNA, and transfected it into mouse erythroleukemia cells. All the G418-resistant clones analyzed acquired and expressed the human beta globin gene. By serial passage of the cells in increasing concentrations of methotrexate, the exogenous human beta globin genes were stably amplified in all lines, and all increased their globin mRNA expression roughly proportional to their augmented copy number. Most of the clones further increased their beta globin expression on addition of an erythroid stimulus (dimethylsulfoxide). These results indicate that globin gene amplification may be useful in increasing globin mRNA expression in further experiments whose goal is gene therapy.
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PMID:Regulated expression of amplified human beta globin genes. 244 79

The current status of gene transfer experiments indicates that it is possible to provide (1) safe and efficient retroviral packaging lines for gene transfer; and (2) vectors containing the human beta-globin genes and selectable marker genes which can be transmitted into erythroid cells and are appropriately expressed. In animal autologous bone marrow transplantation experiments, stable and high-level expression of retroviral vectors containing human beta-globin genes has not yet been achieved. New retroviral vectors are being tested that contain different components of the retrovirus as well as the newly described enhancer elements 5' to the epsilon gene and surrounding the beta-globin gene. The long-term goal of human gene therapy for sickle cell disease then consists of constructing optimally safe and efficient retroviral packaging lines as well as retroviral vectors containing the human beta-globin gene and selectable markers such as the neoR gene. One would then remove bone marrow cells from patients with sickle cell disease, transfer the retroviral vectors into the bone marrow cells, and subject the cells to G418 selection in vitro. Next, one would ablate the host bone marrow and autotransplant the manipulated bone marrow bearing the retroviral vector. Finally, one would analyze the reconstituted bone marrow for human beta-globin gene expression. These experiments must await the demonstration of safe and efficient gene transfer in animals, and particularly experiments must be done in monkeys prior to the use of these approaches in humans. Alternative approaches to gene therapy include direct correction of the defect in the beta-globin gene by site-specific recombination of the defective gene with incoming normal gene sequences. This technology is not yet achievable, although preliminary experiments have been performed in which gene correction at a low frequency has been obtained. The obvious advantage of this approach is that the native human beta gene would be reconstituted. Site-specific recombination and addition of normal beta genes to the human genome represent exciting and feasible approaches to human gene therapy using the extraordinary resources of modern molecular and cellular biology. Success in treating disorders of human hemoglobin only awaits additional technical advances for increasing the efficiency of gene transfer and the level of gene expression.
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PMID:Gene transfer. A potential approach to gene therapy for sickle cell disease. 267 70

Human globin genes can be transferred into mouse and human erythroid cells in culture, and can be appropriately expressed at the mRNA level in these cells. A plasmid containing a human beta globin gene is expressed in mouse erythroleukemia cells (MELC), and another containing a human epsilon or gamma gene is expressed in human erythroleukemia (K562) cells. A neomycin resistance (neoR) gene on the plasmids has been used to select for those cells containing the transferred globin genes; this selection may favor the expression of the globin genes by providing chromosomal positions requiring neoR expression. Analyzing clones resistant to G418, a neomycin analogue, demonstrated globin mRNA expression and induction. Retroviral vectors have also been used to transfer and appropriately express human beta genes in MELC. In addition, a plasmid containing a dihydrofolate reductase (DHFR) gene as well as neoR and beta globin genes has been used to amplify and express beta globin mRNA in MELC. These experiments suggest that high level appropriate expression of human beta globin genes is feasible and provides potentially useful approaches to the long-range goal of gene therapy for sickle cell anemia and beta thalassemia.
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PMID:Human globin gene expression after gene transfer. 347 10

Gene therapy approaches for beta-thalassemia and sickle cell anemia focus on the transfer of a human beta-globin gene into the patient's hematopoietic stem cells (HSC). Expression of the transferred sequences should be erythroid specific and match the expression of the endogenous alpha-globin genes in adult erythropoiesis. Here we explore the potential of recombinant adeno-associated virus (AAV) vectors for human beta-globin gene transfer. We have constructed a recombinant AAV-vector containing a human beta-globin gene together with the DNasel hypersensitive sites 4, 3 and 2 of the human beta-globin locus control region. The vector replicates to high titers and can efficiently transduce hematopoietic and non-hematopoietic cells. In transduced and G418 selected murine erythroleukemia (MEL) cell clones, human beta-globin gene expression was regulated and reached levels comparable to endogenous murine beta maj. These data show that AAV-vectors are promising tools in gene therapy approaches for the haemoglobinopathies.
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PMID:Regulated high-level human beta-globin gene expression in erythroid cells following recombinant adeno-associated virus-mediated gene transfer. 767 Nov 9

We describe a two-step strategy to alter any mouse locus repeatedly and efficiently by direct positive selection. Using conventional targeting for the first step, a functional neo gene and a nonfunctional HPRT minigene (the "socket") are introduced into the genome of HPRT- embryonic stem (ES) cells close to the chosen locus, in this case the beta-globin locus. For the second step, a targeting construct (the "plug") that recombines homologously with the integrated socket and supplies the remaining portion of the HPRT minigene is used; this homologous recombination generates a functional HPRT gene and makes the ES cells hypoxanthine-aminopterin-thymidine resistant. At the same time, the plug provides DNA sequences that recombine homologously with sequences in the target locus and modifies them in the desired manner; the plug is designed so that correctly targeted cells also lose the neo gene and become G418 sensitive. We have used two different plugs to make alterations in the mouse beta-globin locus starting with the same socket-containing ES cell line. One plug deleted 20 kb of DNA containing the two adult beta-globin genes. The other replaced the same region with the human beta-globin gene containing the mutation responsible for sickle cell anemia.
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PMID:Deletion and replacement of the mouse adult beta-globin genes by a "plug and socket" repeated targeting strategy. 793 10