Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UMLS:C0039730 (thalassemia)
 10,305 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Over the past five years, several new defects in the beta-thalassemias have been described from this laboratory using both restriction enzyme and sequencing analyses of cloned beta-thalassemia genes. The enzyme HphI has been shown to recognize a single nucleotide change at the 5' end of beta-IVS 2, and, using restriction enzyme analysis, demonstrated for the first time a specific defect associated with beta(0)-thalassemia. Cloning and sequencing of a beta-thalassemia gene have identified a single base change within IVS 2 at a position 705 nucleotides from the 5' end of IVS 2 that results in a beta(0)-thalassemia phenotype; no normal splicing occurs in this gene despite the fact that both the 5' and 3' ends of IVS 2 are unchanged. A unique and strong cryptic 3' acceptor splice site present in the normal gene at a position 580 nucleotides from the 5' end is used extensively in the mutant gene. Studies of this gene have indicated that there are sequences within IVS that are responsible for optimal expression of this gene; changes in these sequences can lead to markedly abnormal patterns of splicing. In addition, beta-globin gene expression has been evaluated in human erythroleukemia cells, K562 cells, and, although stable transformants with integrated beta-globin genes have been obtained, none of these transformants expressed the added beta-globin genes. This is presumably due to trans-acting factors or distal cis-acting effects that suppress the expression of these added beta-globin genes. In addition, a low epsilon-producing cell line, Bos cells, was used as a recipient for an exogenous epsilon-globin gene. A neomycin resistance gene was cotransfected into these cells, and a neomycin analogue (G418) was used to select cells containing both the neomycin resistance and epsilon-globin genes. Using Southern blotting, 10 of 11 stably transformed G418-resistant lines, which contain intact epsilon-globin genes, express epsilon-globin mRNA at much higher levels than the Bos cells into which they were transfected. Two of these lines express the epsilon-globin genes at a level comparable to that of wild-type K562 cells. These results indicate that the transfer and expression of human globin genes in human erythroid cells is feasible, and can occur at a high level.(ABSTRACT TRUNCATED AT 400 WORDS)
 ...
PMID:Abnormal globin gene structure and expression in beta-thalassemia. 299 Feb 98

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.
 ...
PMID:Regulated high-level human beta-globin gene expression in erythroid cells following recombinant adeno-associated virus-mediated gene transfer. 767 Nov 9

We sought to investigate the usefulness of the adeno-associated virus 2 (AAV)-based vectors to suppress the excess production of the human alpha-globin gene product towards developing a treatment modality for beta-thalassemia since accumulation of free alpha-globin reduces the lifespan of red blood cells in these patients. We constructed recombinant AAV virions containing the human alpha-globin gene sequences in antisense orientation driven by the herpesvirus thymidine kinase (TK) promoter, the SV40 early gene promoter, and the human alpha-globin gene promoter, respectively, as well as a bacterial gene for resistance to neomycin (neoR) as a selectable marker. These recombinant virions were used to infect a human erythroleukemia cell line (K562) that express high levels of alpha-globin mRNA. Clonal populations of neoR cells were obtained after selection with the drug G418, a neomycin analogue. Total genomic DNA samples isolated from these cells were analyzed on Southern blots to document stable integration of the transduced neo and alpha-globin genes. Total cellular RNA samples isolated from mock-infected and recombinant virus-infected cultures were also analyzed by Northern blots. Whereas the TK promoter-driven antisense alpha-globin sequences showed no inhibition of expression of the endogenous alpha-globin gene, the SV40 promoter and the alpha-globin gene promoter-driven antisense alpha-globin sequences suppressed the expression of this constitutively over-expressed gene by approximately 29 and 91%, respectively, at the transcriptional level. These studies suggest the feasibility of utilizing the AAV-based antisense gene transfer approach in the potential treatment of beta-thalassemia.
 ...
PMID:Suppression of human alpha-globin gene expression mediated by the recombinant adeno-associated virus 2-based antisense vectors. 829 80

Trials of retroviral vector-mediated human beta-globin gene transfer were hampered by low titers, unstable vector transmission, and low-level expression of transferred gene. With the goal of optimizing the retrovirally encoded human beta-globin gene expression cassette for gene therapy of beta-thalassemia, we generated 3 series of vector constructs (a total of 12 constructs) and investigated the effects of the proximal promoter, 3' - enhancer, and derivatives from the beta-locus control region or alpha-major regulatory element on virus titer, vector transmission stability, and gene expression. The virus titers for 9 of the 12 vector constructs ranged between 2.8 x 10(4) cfu/mL and 1.0 x 10(6) cfu/mL. We found that proviral DNA was intact in most G418- resistant murine erythroleukemia (MEL) cell clones for 5 vector constructs, while obvious genetic instability was observed for 4 other vector constructs. MEL cells harboring the intact provirus were induced to differentiate, and human beta-globin gene expression was analyzed with RNase protection assay. The percentage of human beta-globin transcript relative to endogenous murine alpha-globin transcript were 101.8 +/- 64.3% (n = 10), 40.1 +/- 28.7% (n = 4), 31.1 +/- 31.9% (n = 12), 52.4 +/- 11.2% (n = 12), and 53.6 +/- 8.6% (n = 12) for the 5 constructs, respectively, demonstrating the development of optimized retroviral vectors for beta-globin gene therapy with murine erythroid cell lines as a model. Unexpectedly, we also documented that the point mutation 8700(C-->T) in DNase I hypersensitive site 2 (HS2) core fragment might contribute to low-level expression of the human beta-globin gene, based on a comparison of results from transfected and transduced MEL cells and sequence analysis of proviral DNA.
 ...
PMID:Evaluation of optimal expression cassette in retrovirus vector for beta-thalassemia gene therapy. 1274 54

Nonsense mutations, giving rise to UAA, UGA and UAG stop codons within the coding region of mRNAs, promote premature translational termination and are the leading cause of approx. 30% of inherited diseases, including cystic fibrosis, Duchenne muscular dystrophy and thalassaemia. For instance, in beta(0)39-thalassaemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well-described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, aminoglycoside antibiotics have been tested on mRNAs carrying premature stop codons. These drugs decrease the accuracy in the codon-anticodon base-pairing, inducing a ribosomal read-through of the premature termination codons. Interestingly, recent papers have described drugs designed and produced for suppressing premature translational termination, inducing a ribosomal read-through of premature but not normal termination codons. These findings have introduced new hopes for the development of a pharmacological approach to the therapy of beta(0)39-thalassaemia. In this context, we started the development of a cellular model of the beta(0)39-thalassaemia mutation that could be used for the screening of a high number of aminoglycosides and analogous molecules. To this aim, we produced a lentiviral construct containing the beta(0)39-thalassaemia globin gene under a minimal LCR (locus control region) control and used this construct for the transduction of K562 cells, subsequently subcloned, with the purpose to obtain several K562 clones with different integration copies of the construct. These clones were then treated with Geneticin (also known as G418) and other aminoglycosides and the production of beta-globin was analysed by FACS analysis. The results obtained suggest that this experimental system is suitable for the characterization of correction of the beta(0)39-globin mutation causing beta-thalassaemia.
 ...
PMID:Development of K562 cell clones expressing beta-globin mRNA carrying the beta039 thalassaemia mutation for the screening of correctors of stop-codon mutations. 1921 18

In several types of thalassemia (including beta(0)39-thalassemia), stop codon mutations lead to premature translation termination and to mRNA destabilization through nonsense-mediated decay. Drugs (for instance aminoglycosides) can be designed to suppress premature termination, inducing a ribosomal readthrough. These findings have introduced new hopes for the development of a pharmacologic approach to the cure of this disease. However, the effects of aminoglycosides on globin mRNA carrying beta-thalassemia stop mutations have not yet been investigated. In this study, we have used a lentiviral construct containing the beta(0)39-thalassemia globin gene under control of the beta-globin promoter and a LCR cassette. We demonstrated by fluorescence-activated cell sorting (FACS) analysis the production of beta-globin by K562 cell clones expressing the beta(0)39-thalassemia globin gene and treated with G418. More importantly, after FACS and high-performance liquid chromatography (HPLC) analyses, erythroid precursor cells from beta(0)39-thalassemia patients were demonstrated to be able to produce beta-globin and adult hemoglobin after treatment with G418. This study strongly suggests that ribosomal readthrough should be considered a strategy for developing experimental strategies for the treatment of beta(0)-thalassemia caused by stop codon mutations. Am. J. Hematol., 2009. (c) 2009 Wiley-Liss, Inc.
 ...
PMID:Production of beta-globin and adult hemoglobin following G418 treatment of erythroid precursor cells from homozygous beta(0)39 thalassemia patients. 1981 11