Fig. (1).

In vitro evaluation of LentiGlobin lentiviral vectors. A) Diagram of the LentiGlobin HPV569 and BB305 lentiviral vectors. The 3′ β-globin enhancer, the 372 base pairs (bp) IVS2 deletion in intron 2 (triangle), the β^A-T87Q mutation (ACA [Thr] to CAG [Gln]) and DNase I hypersensitive sites (HS) 2, HS3, and HS4 of the human β-globin locus control region (LCR) are indicated. Safety modifications including the 400 bp deletion in the U3 of the right HIV LTR, the rabbit β-globin polyA signal and the 2 × 250 bp cHS4 chromatin insulators are indicated. βp, human β-globin promoter; cPPT/flap, central polypurine tract; HIV LTR, human immunodeficiency type-1 virus long-terminal repeat; ppt, polypurine tract; RRE, Rev-responsive element; Ψ+, packaging signal. B) Functional (transducing unit/mL) and physical (ng p24/mL) titers of lentiviral vectors produced in HEK293T cells, before (harvest) and after purification/concentration (final). C) Y axis: Vector copies = Average vector copy number per cell in transduced human CD34+ cells. D) Y axis: Vector copies = Average vector copy numbers per cell in transduced progenitor cells, using either short-term cultures to determine colony forming cells (CFCs) or long-term cultures to determine long-term culture initiating cells (LTC-ICs). MOI was 50. E) HPLC profile of globin chains from pooled erythroid colonies obtained from HPV569 (top) and BB305 (bottom) lentiviral vector-transduced CD34+ cells. F) Y axis: β^A-T87Q globin chains as a percentage of all non-α globin chains as detected by HPLC.