Vector constructs and gp91^phox expression in PLB-XCGD cells. (a) Schema of the self-inactivating (SIN) alpharetroviral (SIN-α) and SIN lentiviral (SIN-LV) proviruses used in this study. The transgene cassette contains the elongation factor-1α promoter in its intronless, 240 bp short version (EFS) promoter and the codon-optimized gp91s and is followed by a woodchuck hepatitis virus posttranscriptional regulatory element (oPRE). In addition, indicated are splice donor and acceptor site (SD and SA), Rev-responsive element (RRE), central polypurine tract (cPPT) and PPT, the direct repeat element (DRE) for the alpharetroviral provirus and remaining viral gag-coding sequences (Δgag) for the lentiviral construct. (b) Human myelomonocytic PLB-XCGD cells transduced with either SIN alpharetroviral vector (alpha) or its SIN lentiviral (LV) analogue were subjected to comparative expression analysis by flow cytometry (n = 3; mean + SD). Transduced populations were analyzed for MFI in the gp91^phox positive subfraction (MFI (gp91⁺)) undifferentiated (undiff; CD11b^-) and after 8 days of myeloid differentiation (diff; CD11b⁺) in parallel. Mean percentages of gp91^phox positive cells upon differentiation are indicated below the corresponding bar's label. (c) Comparative analysis of effective transcription on proviral level by a SIN alpharetro- or equivalent lentiviral vector. Transduced cell populations (n = 4) with varying percentage of gp91^phox expressing cells were FACS-sorted into gp91^phox positive and negative populations, and subsequently analyzed for vector copy numbers (VCN). To estimate the fraction of transcriptionally active proviral integrations, the VCN of the fraction expressing gp91^phox was set in relation to the corresponding cumulative VCN of positive and negative fractions. (d) PLB-XCGD cells were transduced (td) with the VSV-G pseudotyped AS.EFS.gp91s vector at different multiplicity of infection (MOI) as indicated. Percentage of gp91^phox positive cells was determined 5 days post transduction (ptd) by flow cytometry. Representative blots from flow cytometric analysis of transduced PLB-XCGD cells. Mean fluorescence intensities (MFI) of gp91^phox positive populations were as indicated. Stained nontransduced (ntd) PLB-XCGD and PLB-985 wild-type (wt) cells served as controls. Mean VCNs were determined at day 7 after transduction by qPCR. (e) Clonal populations (n = 32) of AS.EFS.gp91s transduced cells established by limited dilution were grouped according to VCN. Expression levels compared to PLB-XCGD control were determined as ΔMFI (mean + SD) by flow cytometry of either undifferentiated cells (undiff; CD11b⁻) or cells subjected to 8 days of differentiation (diff; CD11b⁺).

Long-term analysis of gp91^phox expression and functional evaluation in PLB-XCGD cells. X-linked chronic granulomatous disease (X-CGD) PLB-985 cells were transduced with AS.EFS.gp91s at a MOI of 0.1 and transgene expressing cells were enriched by magnetic sorting. (a) Long-term analysis of gp91^phox expression levels (MFI) in sorted cells (filled symbols) as compared with that of wild-type PLB-985 cells (open symbols). (b) Percentage of gp91^phox positive cells in the sorted population over time. (c) Cytochrome C assay comparing superoxide production per minute in transduced cells 5 weeks (ST) and 20 weeks (LT) after transduction. Superoxide production was compared with that of wild-type cells after myeloid differentiation. Three individually differentiated populations were analyzed in duplicates. (d) Kinetics of cytochrome C reduction determining superoxide production upon PMA stimulation after 8 days of myeloid differentiation of PLB-XCGD ntd, PLB-985 and sorted PLB-XCGD td AS.EFS.gp91s or LV.EFS.gp91s as indicated. Error bars = mean + SD. PMA, phorbol myristate acetate.

Analysis of aberrant transcripts induced by self-inactivating (SIN) alpharetroviral or SIN lentiviral vectors by RT-PCR and 5′RACE. (a and b) Monoclonal PLB-XCGD populations harboring either SIN-α or SIN-lentiviral (LV) integrations in intronic genomic sequences were subjected to RT-PCR for detection of aberrantly spliced transcripts. Schematical representation of proviral intronic integrations in sense orientation to the affected gene for SIN-α (a) or SIN-LV (b). Splice donor (SD) and acceptor sites (SA) mentioned in are indicated. The internal promoter is indicated by a thick black arrow whereas primers used for RT-PCR are drawn as narrowed black arrows (compare Supplementary Table S1 online). The open arrow heads denote the primers used for positive control on gDNA together with the alpharetroviral primers used for detection of cellular exon–vector fusion transcripts (compare Supplementary Figure S2c online). Cryptic splice sites as defined in silico by the NetGene2 software are shown above (sense orientation) or below (reverse orientation) of the proviral schemes. (b) Schematic representation of the aberrant splice events between the cellular exon and the vector sequence are shown below the lentiviral provirus. The sequence of the retrieved fusion transcripts is shown at the right (sequence centered at the exon–vector fusion ± 10 bp). Exon sequences are in black, vector sequences are shown in white lettering. Transcript labels according to Supplementary Figure S2d online. (c) RNA of polyclonal PLB-XCGD cells transduced with either AS.EFS.gp91s or LV.EFS.gp91s and positively sorted for gp91^phox expression was subjected to 5′RACE. Leader specific primers (alpha, LV) were used according to the respective vector transduced. RNA derived from nontransduced PLB-XCGD served as negative control.

Transduction of murine X-linked chronic granulomatous disease (X-CGD) lineage negative bone marrow (BM) cells and nitroblue tetrazolium (NBT) assay. (a) Lin⁻ BM cells from gp91^phox-deficient mice were transduced 2 days after isolation with an MOI of 15 and analyzed for gp91^phox expression 5 days after transduction by flow cytometry. (b) Individually transduced cell populations (n = 4; mean + SD) were differentiated toward myeloid colony forming units (CFU) in semi-solid methylcellulose in duplicates. After 10 days functional analysis for ROS production was performed by a NBT assay upon PMA stimulation. Count of NBT positive CFUs as well as total CFUs was determined and set in relation to the total number of colonies in the plates. (c) Genomic DNA was harvested from NBT positive and negative CFUs (as indicated) and subjected to an alpharetro-provirus specific PCR (176 bp) in parallel with a murine β-actin specific reaction (138 bp). Only β-actin positive colonies are shown. PMA, phorbol myristate acetate.

Reconstitution of gp91phox expression and NADPH oxidase activity in vivo. Alpharetrovirally transduced murine X-linked chronic granulomatous disease (X-CGD) Lin⁻ cells were transplanted into SJL recipient mice (n = 5) and analyzed 10 weeks after transplantation for gp91^phox expression by flow cytometry. Genomic DNA was isolated and vector copy numbers (VCN) was determined by qPCR. (a) Percentages of gp91^phox expressing (gp91⁺; black) and oxidase positive (rho123⁺; white) granulocytes (CD11b⁺ Gr-1⁺) in CD45.2⁺ peripheral blood cells are plotted side by side for each individual animal. Expression data was obtained by intracellular FACS staining and oxidase activity was measured in a FACS based dihydrorhodamine assay by rho123 capture in granulocytes upon phorbol myristate acetate (PMA) stimulation. A control without PMA stimulation is shown for animal #2. Mean VCNs are indicated for each animal. (b) The mean fluorescence intensities (MFI) of rho123⁺ myeloid cells (CD45.2⁺ CD11b⁺) are shown for wild-type (n = 2) and alpharetrovirally treated transplanted animals (n = 5). Nontransduced cells form gp91^−/− mice (X-CGD) serve as controls (n = 2). (c) gp91^phox expression in peripheral blood, spleen, and bone marrow subsets for animal #7 (black line) is shown in overlay with the negative X-CGD control (gray). Only CD45.2⁺ cells were considered for this analysis. Granulocytes (Gran; CD11⁺Gr-1⁺), Monocytes (Mono; CD11⁺Gr-1^-), T-cells (CD3e⁺), and B-cells (B220⁺). CD45.2⁺, cells indicate overall expression in donor cells; Lin⁻, Sca1⁻, cKit⁻ (L⁻S⁻K⁻), committed hematopoietic cells; L⁻S⁻K⁺: committed myeloid progenitors; L⁻S⁺K^-: include early committed lymphoid progenitors; L⁻S⁺K⁺: enriched for HSC and multipotent progenitors; PB, peripheral blood.

Phenotypic correction of human primary X-linked chronic granulomatous disease (X-CGD) cells by self-inactivating (SIN) alpharetroviral gp91^phox expression. (a) Human CD34⁺ X-CGD cells were transduced with AS.EFS.gp91s one day after immunomagnetic enrichment. Transgene expression was analyzed in CD34⁺ cells 3 days after transduction by surface staining (FACS). The transduced CD34⁺ cells (black line) are shown in overlay with the negative CD34⁺ X-CGD control (grey). (b) Functional reconstitution of superoxide activity in colony forming cells derived from transduced CD34⁺ cells were plated on methycellulose 2 days after transduction and superoxide activity was determined by the nitroblue tetrazolium (NBT) assay. (c) Transduced CD34⁺ X-CGD (black line) and control cells (X-CGD = light gray; wild-type = dark gray) were cultured in the presence of human granulocyte-colony stimulating factor to induce myeloid differentiation and analyzed for extracellular gp91^phox expression in CD11b⁺ cells. Analysis was performed 10 days after transduction. (d) After 25 days of differentiation (day 28 after transduction) functional activity was assessed by intracellular rhodamine123 (rho123) accumulation upon phorbol myristate acetate (PMA) stimulation by flow cytometry. Percentages and MFI of rho123⁺ cells in the CD11b^high+ subpopulations are indicated. (e) Superoxide production (day 27 after transduction) was also measured by cytochrome C reduction upon PMA stimulation (n = 2). Values for AS.EFS.gp91s transduced cells are given relative to wild-type control (f) Transduced X-CGD CD34⁺ cells as well as control cells were transplanted into NSG mice 2 days after transduction. Eight weeks later CD34⁺ cells were isolated from bone marrow by magnetic sorting and cultivated in differentiating conditions for 3 weeks before analysis for gp91^phox expression by flow cytometry. The viable CD11b⁺ hCD45⁺ subfraction is plotted for X-CGD (light gray), wild-type (dark gray) and transduced X-CGD cells (black line).