Development of a Condensed Locus Control Region Cassette and Testing in Retrovirus Vectors forAγ-Globin,

ABSTRACT: Retrovirus vectors forAγ-globin are being developed for the treatment of β chain hemoglobinopathies. Toward the goal of achieving therapeutic expression levels, core elements of the β-globin locus control region (LCR) hypersensitive sites (HS) were screened for enhancer activity in erythroid MEL and K562 cell lines using a drug-resistant colony assay. When used alone, core elements of HS1, HS3, and HS4 showed no activity and a fragment for HS2 showed only modest activity in the colony assay. However, a 1.1 kb combination of fragments for HS2, HS3, and HS4 (termed a nLCR) enhanced colony formation 17-fold in K562 cells and 94-fold in MEL cells. Addition of an HS1 fragment enhanced nLCR activity only modestly in MEL cells. When linked to a β-globin gene, the 1.1 kb nLCR enhanced globin mRNA expression to 82% per copy of mouse α-globin in transfected MEL cells. Inclusion of a nLCR in retrovirus vectors containing a β-globin promoter and variousAγ-globin gene expression cassettes resulted in extreme genetic instability and reduced titers. Specific deletions were abrogated by removing homologous sequences, but random recombinations were still observed at significant frequencies. In MEL cells containing intact provirus,Aγ-globin mRNA produced by an optimal vector containing the nLCR was only 2-fold higher (8.5% vs. 3.9% per copy of mouse α-globin) compared to the same vector without the nLCR. These data suggest that vector elements detract from the ability of the nLCR to enhance expression of the βpr.Aγ cassettes.