Dual-regulated lentiviral vector for gene therapy of X-linked chronic granulomatosis

Abstract

Regulated transgene expression may improve the safety and efficacy of hematopoietic stem cell (HSC) gene therapy. Clinical trials for X-linked chronic granulomatous disease (X-CGD) employing gammaretroviral vectors were limited by insertional oncogenesis or lack of persistent engraftment. Our novel strategy, based on regulated lentiviral vectors (LV), targets gp91(phox) expression to the differentiated myeloid compartment while sparing HSC, to reduce the risk of genotoxicity and potential perturbation of reactive oxygen species levels. Targeting was obtained by a myeloid-specific promoter (MSP) and posttranscriptional, microRNA-mediated regulation. We optimized both components in human bone marrow (BM) HSC and their differentiated progeny in vitro and in a xenotransplantation model, and generated therapeutic gp91(phox) expressing LVs for CGD gene therapy. All vectors restored gp91(phox) expression and function in human X-CGD myeloid cell lines, primary monocytes, and differentiated myeloid cells. While unregulated LVs ectopically expressed gp91(phox) in CD34(+) cells, transcriptionally and posttranscriptionally regulated LVs substantially reduced this off-target expression. X-CGD mice transplanted with transduced HSC restored gp91(phox) expression, and MSP-driven vectors maintained regulation during BM development. Combining transcriptional (SP146.gp91-driven) and posttranscriptional (miR-126-restricted) targeting, we achieved high levels of myeloid-specific transgene expression, entirely sparing the CD34(+) HSC compartment. This dual-targeted LV construct represents a promising candidate for further clinical development.

Figures

Figure 1

Optimized detargeting of the hematopoietic stem/progenitor cells (HSPC) compartment by miRNA target sequences. (a) Measuring miRNA activity in primary human HSPC by bidirectional reporter lentiviral vectors (BdLVs). Comparing the miR-126 reporter, BdLV.126T(2), with a control (Ctrl-)BdLV allows precise quantification of GFP downregulation without the need for matching vector copy number (see Methods section for details). The scheme shows in vitro and in vivo assays to comprehensively measure the activity of 126T(2) in human hematopoiesis. (b) Activity of the 126T(2) target in cord blood (CB) (n = 2 donors) and bone marrow (BM) (n = 4 donors), either in the HSPC compartment (CD34+CD38−CD90+, CD34+38−, and 34+38+, 2 days after transduction), or after 2 weeks in myeloid differentiating condition (G-CSF and FCS 10%). CFU-G/M, colony forming units granulocyte/monocyte, CD34hiCD13+; MB, myeloblasts, CD34+/lowCD13+CD11b−; PMC, promyelocytes, CD34−CD13+CD11b−; MC, myelocytes, CD13+CD11b+CD16−; MMC/GR, metamyelocytes/granulocytes, CD13+CD11b+CD16+. The dotted line represents the GFP mean fluorescence intensity (MFI) of the Ctrl BdLV. The differences in GFP expression between the subsets are highly significant (Two-way analysis of variance (ANOVA)), while there is no significant difference between HSPC source (CB versus BM). (c,d) Activity of 126T(2) in vivo. BM CD34+ HSPC were transduced with BdLV.126T(2) or Ctrl.BdLV, and transplanted into NSG mice (n = 4 per group). (c) Representative FACS plots show reporter expression in human CD45+ cells recovered from the BM of hematochimeric mice at 24 weeks post-transplantation. Upper row: plots gated on CD34+CD38− HSPC; lower row: FACS plots gated on CD33+CD11b+ myeloid progeny. (d) Quantification of the in vivo activity of the 126T(2) in the indicated subpopulations. The following surface marker profiles were used to define subpopulations: HSC/MPP: CD34+CD38−CD45RA−CD10−; MLP: CD34+CD38−CD45RA+CD10+ or - as indicated; CMP/MEP: CD34+CD38+CD45RA−; GMP: CD34+CD38+CD45RA+CD10−; B/NK: CD34+CD38+CD45RA+CD10+. The dotted line represents the GFP MFI of the Ctrl BdLV. Statistics were performed by one-way ANOVA, and * indicates P < 0.05 in Bonferroni's post test against CD33+ cells. FACS, fluorescence-activated cell sorting; FCS, fetal calf serum; GFP, green fluorescent protein; PGK, phosphoglycerate kinase.

Figure 2

Transcriptional targeting by the SP146/gp91phoxpromoter in human BM populations. (a) Vector maps of the myeloid-specific promoter (MSP) based on a minimal gp91phox promoter from the endogenous locus fused to the synthetic SP146 element and the ubiquitous PGK promoter (Ctrl LV) which was used as a reference. (b) CD34+ bone marrow (BM) cells (n = 4, two different donors) were transduced with the vectors indicated in (a), and vector copy number (VCN) was assessed after 14 days of in vitro culture (mean + SEM). (c) Transgene expression during in vitro culture is shown for the two vector groups (mean + SEM). Culture conditions and subpopulations are the same as in Figure 1d. P < 0.001 (two-way analysis of variance (ANOVA)), * indicates significance levels after Bonferroni post-test correction, with the following reference populations: before the slash: MSP versus PGK; after the slash: MSP versus MSP.126T. (d) Representative FACS plots showing GFP expression of MSP- (black line) or Ctrl LV- (gray area) transduced cells in CD34+CD38+CD90+ hematopoietic stem/progenitor cells (HSPC) (top) or CD34−CD13+CD11b+ myeloid progeny (bottom). (e) Mean fluorescence intensity (MFI) (arithmetic mean) of the GFP-positive cells shown in (c). P < 0.001 (two-way ANOVA), and * indicates significance levels after Bonferroni post-test correction. (f) Representative histograms and MFI quantification in the indicated subpopulations recovered from day 14 myeloerythroid colonies (n = 3). Two-way ANOVA: P < 0.0001 (subpopulations), ns (MSP versus MSP.126T). (g) Differentiation-stage-specific activity of PGK, PGK.126T(2), MSP, MSP.126T(2). Indicated subpopulations (see Figure 1b for legend) were identified in a 2-week myeloid differentiation culture of CD34+ bone marrow (BM), and MFI of each subpopulation was internally normalized to the most primitive cell fraction in the culture, i.e., CD34hi13−11b− cells (HSPC). (h,i) In vivo activity of the MSPs. BM CD34+ HSPC were transduced with MSP LV, MSP.LV.126T(2), or PGK Ctrl.LV, and transplanted into NSG mice (n = 4 per group). Representative FACS plots show GFP expression in the indicated subpopulations of human CD45+ cells recovered from the PB of hematochimeric mice at 10 (CD33+, CD19+ cells) or 17 weeks (CD3+ cells) post-transplantation. The bar graph on the right shows GFP expression of the MSP in the indicated subpopulations (green bars, median; d) and the MFI of the GFP-positive cells relative to the mean MFI of the Ctrl LV in the indicated subpopulation (white bars, mean + SEM). The dotted line represents the average %GFP+ cells in the CtrlLV group (left y-axis) and the GFP MFI of the Ctrl LV (right y-axis). FACS, fluorescence-activated cell sorting; PB, peripheral blood; PGK, phosphoglycerate kinase.

Figure 3

Prevention of ectopic expression in human CD34+cells of healthy donor transduced with regulated vectors. (a) The histograms represent % of gp91phox positive cells (mean ± SEM) within CD34+ cell gates, in four independent experiments. Statistical analysis was performed with one-way analysis of variance (ANOVA) with Bonferroni post-test correction, * indicates P < 0.05, **indicates P < 0.01, and *** indicates P < 0.001. (b) gp91phox-estimated mean fluorescence intensity (MFI) (as reported in Supplementary Methods) of HD CD34+ cells transduced with the indicated vectors. Statistical analysis was performed as in (a). (c) FACS analysis of transgene expression in human progenitor cell subsets: hematopoietic stem cell (CD34+ CD38− CD90+ CD45RA−), MPP (CD34+ CD38− CD90− CD45RA−), MLP (CD34+ CD38− CD90− CD45RA+), GMP (CD34+ CD38+ CD90− CD45RA+), CMP/MEP (CD34+ CD38+ CD90− CD45RA+). The histograms represent the percentage of gp91phox positive cells in the indicated subsets (n = 3, mean ± SEM). The mean vector copy number ± SEM was 1.0 ± 0.2 for MSP.gp91 and 1.1 ± 0.2 for MSP.gp91.1262T. Statistical analysis was performed with one-way ANOVA with Bonferroni post-test correction, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001. FACS, fluorescence-activated cell sorting; HD, healthy donor; PGK, phosphoglycerate kinase.

Figure 4

Regulated transgene expression with restored gp91phoxexpression and function in X-linked chronic granulomatous disease (X-CGD) cells. (a,b) X-CGD CD34+ cells from Pt1 and Pt2 were transduced with the indicated vectors at 100 MOI and analyzed for the surface expression of gp91phox by FACS, after 4 days of liquid culture in serum-free medium with SCF, FLT3L, TPO, and IL-3. Vector copy number reported for each sample is calculated on the total population after in vitro culture. In (c) bars show the gp91phox MFI of transduced CD34+ cells relative to the mean fluorescence intensity of HD untransduced cells in CD16+CD11b+ and CD14+CD11b+ gated cells for Pt 1 and Pt 2. (d,e) Nicotinamide adenine dinucleotide phosphate oxidase activity in myeloid (CD11b+) cells from Pt1 and Pt2 measured by DHR assay. The bars show the percentage of Rhodamine123 cells after PMA stimulation. DHR, dihydrorhodamine 123; FACS, fluorescence-activated cell sorting; HD, healthy donor; MOI, multiplicity of infection; PMA, phorbol 12-myristate 13-acetate; SCF, stem cell factor; TPO, thrombopoietin.

Figure 5

Restored gp91phoxexpression and function in primary X-linked chronic granulomatous disease macrophages. PB monocytes from three patients were cultured with M-CSF and transduced after 1 week with the indicated vectors in the presence of virus-like particles-VPX. (a) Mean fluorescence intensity and % of gp91phox expression are indicated in cells gated for CD206 and CD14. Untransduced cells and HD macrophages are shown for comparison. (b) Representative NADPH oxidase activity measured by cytochrome c assay is shown. The bars show the superoxide production after PMA-stimulation of patient 1. M-CSF, macrophage colony stimulating factor; PB, peripheral blood; PMA, phorbol 12-myristate 13-acetate; VPX, virion protein X.

Figure 6

Restored gp91phoxexpression and biochemical correction in X-linked chronic granulomatous disease (X-CGD) mice treated with gene-corrected Lin-negative cells. X-CGD mice were irradiated at 900 RAD and injected with X-CGD Lin− cells transduced with the indicated regulated vectors. Mice were sacrificed after 20 weeks and analyzed for gp91phox expression in PB and bone marrow (BM); nicotinamide adenine dinucleotide phosphate oxidase function was evaluated in PB granulocytes and monocytes. (a) Expression of gp91phox in PB Granulocytes (CD11b+, Gr1+, CD48+), monocytes (CD11b+, Gr1−, CD48hi), B cells (B220+, CD48+), and T cells (CD3+). Statistical analysis was performed with one-way analysis of variance with Bonferroni post-test correction, * indicates P < 0.05, ** indicates P < 0.01, and *** P < 0.001. (b) Representative plots of DHR assay on PB cells of mice transplanted with X-CGD Lin- or wt Lin- or or transduced X-CGD Lin−. (c) Mean fluorescence intensity of DHR-positive cells in PB granulocytes and monocytes. (d) The presence of the therapeutic gene gp91phox was investigated in different BM progenitor cell subsets. Statistical analysis was performed as in (a). DHR, dihydrorhodamine 123.