Volume 15, Issue 10, Pages 1863-1871 (October 2007) Improved Human β-globin Expression from Self-inactivating Lentiviral Vectors Carrying the Chicken Hypersensitive Site-4 (cHS4) Insulator Element  Paritha I Arumugam, Jessica Scholes, Natalya Perelman, Ping Xia, Jiing-Kuan Yee, Punam Malik  Molecular Therapy  Volume 15, Issue 10, Pages 1863-1871 (October 2007) DOI: 10.1038/sj.mt.6300259 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Vector design. (a) The BG vector was constructed using a self-inactivating (SIN) lentiviral backbone containing the human β-globin (hβ-globin) gene and promoter and the locus control region (LCR) consisting of hypersensitive sites (HSs) 2, 3, and 4 cloned in the reverse orientation to the viral transcriptional unit, downstream of the rev response element (RRE) and the central polypurine tract (cPPT). (b) The BG-I vector was designed with a 1.2-kb chicken HS4 (cHS4) insulator element inserted to replace the U3 promoter/enhancer deletion (U3Δ). Upon proviral integration into host genome, the U3 region containing the 1.2-kb cHS4 is copied over to the 5′ long terminal repeat. Two additional vectors, (c) BGM and (d) BGM-I, were constructed that carried the mutant P140K methyl guanine methyl transferase (MGMTP140K) complementary DNA in a direction opposite to the hβ-globin gene, under the control of human phosphoglycerate kinase promoter. Proviral forms of all vectors are shown. Molecular Therapy 2007 15, 1863-1871DOI: (10.1038/sj.mt.6300259) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 Human β-globin (hβ-globin) expression from BGM and BGM-I murine erythroleukemia (MEL) clones after β-chloro-nitrosourea compound selection. (a) Representative flow cytometry histograms of selected BGM and BGM-I clones, labeled with antibody to hβ-globin (filled histograms) overlaid with the fluorescence of similarly labeled un-transduced MEL clones (clear histograms). The percentage hβ-globin expression is indicated at the top right of the histogram overlays. (b) Cumulative data on the hβ-globin-expressing cells in BGM (open circles) and BGM-I clones (closed circles). (c) Coefficient of variation (CV) of hβ-globin expression of the BGM and BGM-I drug-selected MEL clones. The means are represented with a horizontal line and the mean ± SEM and P-values are denoted in the figure. Molecular Therapy 2007 15, 1863-1871DOI: (10.1038/sj.mt.6300259) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Human β-globin (hβ-globin) expression from BGM and BGM-I murine erythroleukemia (MEL) clones without selection. (a) Proportion of hβ-globin-expressing cells/clone transduced with the BGM vector (open circles) and BGM-I (filled circles). Clones were screened for transduction by polymerase chain reaction. (b) Coefficient of variation and (c) mean fluorescence intensity (MFI) of hβ-globin expression/clone in unselected BGM and BGM-MEL clones. Molecular Therapy 2007 15, 1863-1871DOI: (10.1038/sj.mt.6300259) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 Human β-globin (hβ-globin) expression from vectors carrying no drug selection cassette, BG and BG-I. (a) Representative fluorescence-activated cell sorting histograms of BG and BG-I single-integrant clones (filled histograms), overlaid with the fluorescence of un-transduced murine erythroleukemia (MEL) clones (clear histograms). The proportion of hβ-globin-expressing cells is shown on the x-axis and values are indicated at the top right of the histogram overlays. (b) Proportion of hβ-globin expressing cells/clone. (c) The coefficient of variation (CV) of the hβ-globin expression in BG (empty squares) and BG-I (filled squares) transduced MEL clones. Clones were screened for vector integrants by polymerase chain reaction. Mean ± SEM and P-values are shown in the figure. Molecular Therapy 2007 15, 1863-1871DOI: (10.1038/sj.mt.6300259) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 Human β-globin (hβ-globin) messenger RNA (mRNA) expression from murine erythroleukemia (MEL) clones transduced with insulated and un-insulated vectors. (a) A representative ribonuclease protection assay showing hβ-globin mRNA expression in unselected BGM, BGM-I, and un-transduced MEL clones. Murine α-globin expression served as the internal loading control, against which hβ-globin expression was normalized. Quantification of hβ-globin mRNA (percentage hβ/total mα) by phosphorimaging in (b) unselected BGM (open bar) and BGM-I (hatched bar) transduced MEL clones, (c) selected BGM and BGM-I MEL clones, and (d) BG (open bar) and BG-I (checkered bar) MEL clones. Mean ± SEM and P-values are shown in the figure. Molecular Therapy 2007 15, 1863-1871DOI: (10.1038/sj.mt.6300259) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 6 Consistent and higher human β-globin (hβ-globin) expression in vivo from murine hematopoietic stem cells. (a) Representative fluorescence-activated cell sorting histograms of BGM and BGM-I group mice showing hβ-globin expression in peripheral blood red blood cells (RBCs) 5 months after bone marrow transplant. Insets show the same data as a dot-plot, demonstrating the distinct separation of hβ-globin-expressing cells even when present in a small proportion. (b) Cumulative data on the percentage of hβ-globin-expressing RBCs in BGM (open bar) and BGM-I (hatched bar) transplanted mice (n = 12). (c) The coefficient of variation (CV) of hβ-globin-expressing RBCs in vector transplanted mice. (d) Cellulose acetate electrophoresis of peripheral blood lysates at 4 months after transplantation showing mouse hemoglobin tetramers and a chimeric hemoglobin band composed of mα- and hβ-globin chains. (e) Quantification of the chimeric hemoglobin bands by densitometry in the BGM (open bar) and BGM-I (hatched bar) groups of mice. chim. Hb, chimeric hemoglobin. Molecular Therapy 2007 15, 1863-1871DOI: (10.1038/sj.mt.6300259) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 7 Human β-globin (hβ-globin) expression in the clonal progeny of hematopoietic stem cells in secondary mice. (a) Representative dot-plot analysis of control, BGM, and BGM-I group mice showing hβ-globin expression in single-copy secondary spleen colony-forming units (CFU-Ss). Of note, even low proportions of hβ-globin-expressing cells were distinctly separate from non-expressing cells in each CFU-S. (b) Cumulative data on hβ-globin-expressing single-copy CFU-Ss of BGM (open circles) and BGM-I (closed circles). (c) The coefficient of variation (CV) of hβ-globin-expressing cells/CFU-S of BGM and BGM-I secondary mice. Molecular Therapy 2007 15, 1863-1871DOI: (10.1038/sj.mt.6300259) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions