Biochemical correction of X-CGD by a novel chimeric promoter regulating high levels of transgene expression in myeloid cells

Abstract

X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene encoding the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase catalytic subunit gp91(phox). A recent clinical trial for X-CGD using a spleen focus-forming virus (SFFV)-based γ-retroviral vector has demonstrated clear therapeutic benefits in several patients although complicated by enhancer-mediated mutagenesis and diminution of effectiveness over time due to silencing of the viral long terminal repeat (LTR). To improve safety and efficacy, we have designed a lentiviral vector that directs transgene expression primarily in myeloid cells. To this end, we created a synthetic chimeric promoter that contains binding sites for myeloid transcription factors CAAT box enhancer-binding family proteins (C/EBPs) and PU.1, which are highly expressed during granulocytic differentiation. As predicted, the chimeric promoter regulated higher reporter gene expression in myeloid than in nonmyeloid cells, and in human hematopoietic progenitors upon granulocytic differentiation. In a murine model of stem cell gene therapy for X-CGD, the chimeric vector resulted in high levels of gp91(phox) expression in committed myeloid cells and granulocytes, and restored normal NADPH-oxidase activity. These findings were recapitulated in human neutrophils derived from transduced X-CGD CD34(+) cells in vivo, and suggest that the chimeric promoter will have utility for gene therapy of myeloid lineage disorders such as CGD.

Figure 1

Sequence of the chimeric promoter. Transcription binding sites are underlined.

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-fesGp91_s or pCCLchimGp91_s 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.

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.

Figure 4

The chimeric promoter rescues gp91^phox expression and function in primary X-CGD peripheral blood stem cells (PBSCs). Expression of (a,b) gp91^phox and (c–e) NADPH-oxidase function in pCCLchimGp91_s-transduced human X-CGD PBSCs after neutrophil differentiation. Transduced cells were tested for gp91^phox 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.

Figure 5

The chimeric promoter achieves long-term reconstitution of nicotinamide adenine dinucleotide phosphate (NADPH) activity in X-CGD cells. (a) Expression of gp91^phox in the PB of C57Ly5.1 mice transplanted with pCCLchimGp91_s-transduced X-CGD cells. The bars show the percentage of gp91^phox expressing cells among the T (CD3⁺), B (B220⁺), neutrophil (Gr1^high, cd11b⁺), and monocyte (Gr1^low, cd11b⁺) compartments of the engrafted cells (CD45.2⁺). (b) DHR test in the PB cells of C57Ly5.1 mice transplanted with pCCLchimGp91_s-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 (gp91^phox+ donor granulocytes%) and function (DHR⁺ donor granulocytes %) in PB samples from C57Ly5.1 mice 18–19 weeks after transplant with pCCLchimGp91_s-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 gp91^phox in different lineage negative hematopoietic progenitor subsets derived from donor cells. (D) Expression of gp91^phox in bone marrow granulocytes derived from donor cells. The MFI of the gp91^phox+ 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.

Figure 6

Flow cytometric analyses of the expression of human gp91^phox 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 gp91^phox 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) pCCLchimGp91_s-transduced X-CGD CD34⁺PBSCs. (e) Summary histogram plots showing the percentage of gp91^phox+ 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.