Volume 19, Issue 1, Pages 122-132 (January 2011) Biochemical Correction of X-CGD by a Novel Chimeric Promoter Regulating High Levels of Transgene Expression in Myeloid Cells  Giorgia Santilli, Elena Almarza, Christian Brendel, Uimook Choi, Chiara Beilin, Michael P Blundell, Sneha Haria, Kathryn L Parsley, Christine Kinnon, Harry L Malech, Juan A Bueren, Manuel Grez, Adrian J Thrasher  Molecular Therapy  Volume 19, Issue 1, Pages 122-132 (January 2011) DOI: 10.1038/mt.2010.226 Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Sequence of the chimeric promoter. Transcription binding sites are underlined. Molecular Therapy 2011 19, 122-132DOI: (10.1038/mt.2010.226) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Efficacy of the chimeric promoter in myeloid cells. (a) A representative fluorescence-activated cell-sorting (FACS) plot of pCCLchimGFP- and pCCLSFFVGFP-transduced cell lines. HeLa, 293T, Jurkat, LCL, and PLB985 cells were transduced with the indicated vectors and green fluorescent protein (GFP) expression was analyzed by FACS after 5 days. In the lower panel, the histograms show the GFP MFI normalized for vector copy number in each cell line. (b) Western blot analysis of PU.1 and C/EBPα expression in PLB985 cells induced to differentiate into monocytes [phorbol 12-myristate 13-acetate (PMA) treated] or neutrophils [dimethyl formamide (DMF) treated]. Actin is used as loading control. (c) FACS analysis of GFP expression in PLB985 cells transduced with pCCLc-fesGp91s or pCCLchimGp91s after PMA or DMF treatment. (d) Representative FACS overlay plot of GFP expression in human CD34 cells transduced with pCCLchimGFP before and after granulocytic differentiation. The GFP+ percentage and MFI are shown on each plot. MFI, mean fluorescence intensity. Molecular Therapy 2011 19, 122-132DOI: (10.1038/mt.2010.226) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 The chimeric promoter drives a stable and copy number–dependent gene expression. (a) Fluorescence-activated cell-sorting (FACS) analysis of GFP MFI in clonal population over time. After transduction with pCCLchimGFP and pCCLSFFVGFP, PLB985 cells were sorted into single cells. The clones were analyzed for GFP expression at regular intervals. The histogram shows the average GFP MFI for each vector at the indicated time points. The coefficient of variation for each vector is shown in brackets. (b) Histograms show the GFP MFI for each clone in the y axis and the vector copy number in the x axis. The equation of the curve is shown in each plot. CV, coefficient of variation; MFI, mean fluorescence intensity. Molecular Therapy 2011 19, 122-132DOI: (10.1038/mt.2010.226) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 The chimeric promoter rescues gp91phox expression and function in primary X-CGD peripheral blood stem cells (PBSCs). Expression of (a,b) gp91phox and (c–e) NADPH-oxidase function in pCCLchimGp91s-transduced human X-CGD PBSCs after neutrophil differentiation. Transduced cells were tested for gp91phox expression by (a) FACS analysis and (b) by western blot. (a) Average vector copy number (VCN) is shown next to each plot. (c) The upper panel shows a nitroblue tetrazolium test on 96-well plated cells. The lower panel shows the quantification of superoxide production by transduced cells as assessed by the cytochrome c assay. (d) Dihydrorhodamine 123 test. The rhodamine 123-positive cells percentage and MFI are shown next to each FACS plot. MFI, mean fluorescence intensity; WT, wild type; X-CGD, X-linked chronic granulomatous disease. Molecular Therapy 2011 19, 122-132DOI: (10.1038/mt.2010.226) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 The chimeric promoter achieves long-term reconstitution of nicotinamide adenine dinucleotide phosphate (NADPH) activity in X-CGD cells. (a) Expression of gp91phox in the PB of C57Ly5.1 mice transplanted with pCCLchimGp91s-transduced X-CGD cells. The bars show the percentage of gp91phox expressing cells among the T (CD3+), B (B220+), neutrophil (Gr1high, cd11b+), and monocyte (Gr1low, cd11b+) compartments of the engrafted cells (CD45.2+). (b) DHR test in the PB cells of C57Ly5.1 mice transplanted with pCCLchimGp91s-transduced C57Ly5.2 cells. The fluorescence-activated cell-sorting plots show the percentage of DHR+ granulocytes (Gr1, Cd11b+ cells) among the CD45.2+ population. X-CGD and wt C57Ly5.2 cells were used as negative and positive controls, respectively. (c) Analysis of NADPH-oxidase expression (gp91phox+ donor granulocytes%) and function (DHR+ donor granulocytes %) in PB samples from C57Ly5.1 mice 18–19 weeks after transplant with pCCLchimGp91s-transduced cells. One mouse (#2) was found dead. (d) C57 Ly5.1 mice transplanted with transduced X-CGD-lineage negative cells were killed 6 months post-transplantation. Bone marrow cells were either stained with CD45.2, Sca-1, cKit, and lineage-panel antibodies (column A–C) or CD45.2, CD11b, and Gr1 (column D). (A–C) Expression of gp91phox in different lineage negative hematopoietic progenitor subsets derived from donor cells. (D) Expression of gp91phox in bone marrow granulocytes derived from donor cells. The MFI of the gp91phox+ fraction of each sample is normalized for the MFI obtained in the X-CGD control sample (MFI fold over control). DHR, dihydrorhodamine 123; MFI, mean fluorescence intensity; PB, peripheral blood; X-CGD, X-linked chronic granulomatous disease. Molecular Therapy 2011 19, 122-132DOI: (10.1038/mt.2010.226) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Flow cytometric analyses of the expression of human gp91phox by human myeloid cells in the bone marrow of NOD/SCID mice that received transplants of human CD34+PBSCs. Shown are representative dot plots of analyses of gp91phox expression in high side scatter human myeloid (CD45+/CD13+) cells engrafted in chimeric bone marrow from (a) NOD/SCID mice that underwent transplantation with 4-day cultured but (b) nontransduced X-CGD CD34+PBSCs, (c) nontransduced normal CD34+PBSCs, and (d) pCCLchimGp91s-transduced X-CGD CD34+PBSCs. (e) Summary histogram plots showing the percentage of gp91phox+ cells (left panel) and the mean fluorescence intensity (right panel) as average of three mice per group. Analyses are gated to include only those cells that label positive for the CD45 and CD13 antigen (R2 gate). HSC, hematopoietic stem cell; NOD, nonobese diabetic; PBSC, peripheral blood stem cell; SCID, severe combined immunodeficiency. Molecular Therapy 2011 19, 122-132DOI: (10.1038/mt.2010.226) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions