Increased HbF levels in erythroid cells derived from normal CD34⁺ cells transduced with γ-globin lentiviral vectors. (A) Top: Schematic of control SIN lentiviral vector encoding GFP expressed from a modified murine stem cell virus LTR promoter. Bottom: Schematic of SIN, erythroid-specific γ-globin lentiviral vectors, using as transcriptional control elements 3.1 kb of sequences from the β-globin LCR and a 130-bp β-globin promoter. This vector also contains 3′-untranslated sequences (UTR) from the native β-globin coding sequence in the presence or absence of a core 400-bp insulator (open rectangle) element inserted into the deleted U3-region of the 3′-LTR referred to as V5m3 or V5m3-400, respectively. Both vector backbones contain the central polypurine track (cPPT) and the rev responsive element (RRE), as indicated, and have an SIN design in which the promoter and enhancer of the HIV U3 region are deleted. (B) Experimental schema with time course of expansion and differentiation phases as indicated. Time points or intervals are indicated for the specific determinations shown at left. (C) γ-Globin mRNA levels (represented as γ/α ratios) are shown as measured by reverse-transcriptase PCR in the various cell populations on days 8 and 11. (D) Hb electrophoresis demonstrating various production of HbF in erythroblasts derived from CD34⁺ cell populations from 4 independent normal donors transduced using mock conditions or with a lentiviral vector encoding GFP (MOI = 5), or human γ-globin encoding vectors V5m3 or V5m3-400 (MOI = 20), respectively. The percentage of HbF as quantified by HPLC analysis and normalized to average vector copy number in bulk cell populations as determined by Southern blot and densitometry analysis is indicated below each lane. (E) Representative PCR analysis for the presence of vector-encoded sequences in genomic DNA isolated from individual colonies derived from CD34⁺ cells transduced with the indicated γ-globin vectors. Size markers (M) shown at right. V indicates positive control DNA for the vector. Numbers above lanes indicate individual samples. (F) Southern blot analysis of genomic DNA, cut with BglII, from bulk cell populations transduced with the indicated lentiviral vectors. Average vector copy number was determined by densitometry relative to a K562 clone that contains a single copy of an integrated GFP-encoding lentiviral vector. (G) The percentage of HbF, relative to total cellular Hb and normalized to vector copy, in the indicated erythroblast populations (± SEM). *P < .004 compared with the mock control (paired t test); n = 3. Vertical lines have been inserted to represent repositioned lanes on the gel images.