High-level beta-globin expression after retroviral transfer of locus activation region-containing human beta-globin gene derivatives into murine erythroleukemia cells.
The locus activation region (LAR) of the human beta-globin-like gene cluster is characterized by a group of four DNase I hypersensitive sites, which arise specifically in erythroid tissues and are required for a normal pattern of beta-globin-like gene expression. The hypersensitive sites are found at positions 6.1, 10.9, 14.7, and 18 kilobase pairs (kbp) 5' of the epsilon-globin gene. Recently functional assays of the LAR that tested determinants for all four hypersensitive sites showed that expression of the human beta-globin gene was increased to normal or near-normal levels in both transgenic mice and erythroid cells. We constructed retroviral vectors with a human beta-globin gene and the determinant for a single hypersensitive site and measured beta-globin gene expression after retroviral infection of murine erythroleukemia cells. Fragments for the hypersensitive sites at -18 or -10.9 kbp increased human beta-globin RNA levels respectively to 35% or 132% of the endogenous mouse beta maj-globin RNA level. In addition, greater expression was also observed for the neomycin phosphotransferase RNA, which was transcribed from the retroviral LTR, showing that the LAR fragments activated expression from a heterologous promoter. In the context of gene-transfer experiments ultimately aimed at gene therapy, our results show that LAR determinants lead to an increased level of human beta-globin RNA expression after retroviral transfer into erythroid cells. But inclusion of LAR determinants in retroviral vectors also entails the potential risk of activating the expression of nonglobin genes in erythroid cells.
Novak
U
U
Fred Hutchinson Cancer Research Center, Seattle, WA 98104.
Harris
E A
EA
Forrester
W
W
Groudine
M
M
Gelinas
R
R
D000818:Animals; D002460:Cell Line; D003850:Deoxyribonuclease I; D005786:Gene Expression Regulation; D005796:Genes; D005914:Globins / Q000235:genetics*; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute; D051379:Mice; D005810:Multigene Family; D011533:Proviruses / Q000235:genetics; D015183:Restriction Mapping; D012190:Retroviridae / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000378:metabolism
D005914:Globins; D003850:Deoxyribonuclease I