( National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China )

Abstract

Murine erythroleukemia (MEL) cell line was used as a model to evaluate the potential value of retroviral construct containing human β-globin express io n cassette in gene therapy of β-thalassemia and to explore possible mechanisms underlying low expression of retrovirally cloned human β-globin gene. A recomb i nant retroviral vector was constructed, which harbored 2.0 kb β-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 huma n β-LCR. The recombinant retroviruses were generated from an established ψ-2 producer cell line, and by transient transfection of amphotropic packaging cell l ine ψ-A, respectively. The integrity of provirus in transduced MEL cells was det ermined using Southern blot, and the expression of transferred human β-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 indiv idual clone as template. The results demonstrated that the average expression of human β-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 α-globin ge ne, in MEL cells transduced with the recombinant retrovirus from transient tran s fection of ψ-A and MEL cells transfected with the construct, respectively. In M EL cells transduced with virus from ψ-2 producer cell line, however, the av erage expression was less than 3%. A point mutation was detected in HS2 of provirus i n MEL cell clone with low expression of human β-globin gene. The possible mech anisms involved in low expression, including position effect, DNA methylation a nd RNA interference are discussed in addition to the point mutation.