DEVELOPMENT OF A CONDENSED LOCUS-CONTROL REGION CASSETTE AND TESTING IN RETROVIRUS VECTORS FOR (A)GAMMA-GLOBIN

Retrovirus vectors for (A)gamma-globin are being developed for the tre atment of beta chain hemoglobinopathies, Toward the goal of achieving therapeutic expression levels, core elements of the beta-globin locus control region (LCR) hypersensitive sites (HS) were screened for enhan cer activity in erythroid MEL and K562 cell lines using a drug-resista nt colony assay. When used alone, core elements of HS1, HS3, and HS4 s howed no activity and a fragment for HS2 showed only modest activity i n 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 R5 62 cells and 94-fold in MEL cells. Addition of an HS1 fragment enhance d nLCR activity only modestly in MEL cells. When linked to a beta-glob in gene, the 1.1 kb nLCR enhanced globin mRNA expression to 82% per co py of mouse alpha-globin in transfected MEL cells. Inclusion of a nLCR in retrovirus vectors containing a beta-globin promoter and various ( A)gamma-globin gene expression cassettes resulted in extreme genetic i nstability and reduced titers. Specific deletions were abrogated by re moving homologous sequences, but random recombinations were still obse rved at significant frequencies. In MEL cells containing intact provir us, (A)gamma-globin mRNA produced by an optimal vector containing the nLCR was only 2-fold higher (8.5% vs. 3.9% per copy of mouse alpha-glo bin) compared to the same vector without the nLCR, These data suggest that vector elements detract from the ability of the nLCR to enhance e xpression of the beta(pr). (A)gamma cassettes. (C) 1998 Academic Press .