Improved γ-globin vector expression using β-globin LCR elements in a lentiviral vector backbone.

(A) Schematic representations of oncoretroviral- and lentiviral-based γ-globin vectors. MSCV-based oncoretroviral vectors express GFP from the viral LTR and contain the different ^Aγ-globin cassettes in reverse orientation. The β-spectrin (−576) and β-globin promoters (−130), along with their respective 5′ untranslated sequences, were fused to the ^Aγ-globin coding sequence at position −3 and +1 relative to the endogenous translational start site. A 255-bp fragment containing the α-globin HS40 enhancer element was placed adjacent to the promoters. The same β-globin promoter, γ-globin coding sequences, and 3′ untranslated and downstream sequences (466 bp) were also placed downstream of β-globin LCR elements containing sites HS4 (756 bp), HS3 (898 bp), and HS2 (374 bp) in both oncoretroviral and HIV-based lentiviral vector backbones. The γ-globin gene contained a 720-bp deletion in the second intron in all of the vectors. The lentiviral vector HS432 β-^Aγ MSCV-GFP also contained an MSCV LTR-driven GFP cassette. The GFP cassette was removed to yield HS432β-^Aγ. d432β-^Aγ contained a 311-bp deletion in HS4 outside of the “core” element. The β-globin enhancer (β 3′ Enh) or the γ-globin 3′ regulatory element (γ 3′ RE) was placed downstream of the γ-globin coding sequences to derive 2 additional lentiviral vectors. (B) Southern blot analysis, using a radiolabeled GFP probe, of DNA from MEL cells transduced with the oncoretroviral vectors MSCV-GFP HS40β^spectrinAγ and MSCV-GFP HS40β^globinAγ and the lentiviral vector HS432β-^Aγ MSCV-GFP. DNA (10 μg) from each GFP⁺ MEL cell pool was digested to release a full- or near full–length proviral fragment. The respective plasmid DNAs for each construct were concurrently digested and run as controls for correct molecular size. (C) FACS analysis for γ-globin expression (PE fluorescence) following erythroid induction of MEL cells transduced with vectors containing the γ-globin expression cassette driven by the 5′ regulatory elements as indicated above each dot plot. The top row of dot plots shows representative results from 2 independent experiments comparing the 2 HS40-based oncoretroviral vectors. The bottom 2 dot plots are representative results from 3 independent experiments comparing the oncoretroviral-based HS40/β-globin promoter vector and lentiviral-based HS432/β-globin promoter vector. The percentage of γ-globin–positive cells relative to all transduced, GFP⁺ cells is indicated for each dot plot. MEL cells transduced with a vector encoding only GFP failed to show staining for γ-globin (data not shown).