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

Murine erythroleukemia (MEL) cell line was used as a model to evaluate the potential value of retroviral construct containing human beta-globin express io n cassette in gene therapy of beta-thalassemia and to explore possible mechanisms underlying low expression of retrovirally cloned human beta-globin gene. A recombinant retroviral vector was constructed, which harbored 2.0 kb beta-globin gene w it h a 374 bp deletion in intervening sequence II coupled with a mini locus control region (miniLCR) composed of DNaseI hypersensitive sites (HS) 2 and 3 from human beta-LCR. The recombinant retroviruses were generated from an established psi-2 producer cell line, and by transient transfection of amphotropic packaging cell l ine psi-A, respectively. The integrity of provirus in transduced MEL cells was determined using Southern blot, and the expression of transferred human beta-globin gene was detected using RNase protection assay. The structure of provirus was further analyzed by sequence analysis of PCR products from genomic DNA of MEL individual clone as template. The results demonstrated that the average expression of human beta-globin gene was (52.4-/+11.2)% (n=12) and (73.8-/+14.3)% (n=12, without copy-number determination), compared with that of endogenous murine alpha-globin ge ne, in MEL cells transduced with the recombinant retrovirus from transient transfection of psi-A and MEL cells transfected with the construct, respectively. In M EL cells transduced with virus from psi-2 producer cell line, however, the average expression was less than 3%. A point mutation was detected in HS2 of provirus i n MEL cell clone with low expression of human beta-globin gene. The possible mechanisms involved in low expression, including position effect, DNA methylation a nd RNA interference are discussed in addition to the point mutation.
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PMID:[The possible mechanisms underlying low expression of human beta-globin gene cloned in a retroviral vector]. 1241 21

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.
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PMID:Evaluation of optimal expression cassette in retrovirus vector for beta-thalassemia gene therapy. 1274 54