Figure 1.
The efficiency of FOXP3 expression by lentiviral vectors. (A) Vector maps showing the design of the 4 vectors and their mock counterparts within the pCCL backbone. Two bidirectional vectors: LNGFRp-eFOXP3 expressing FOXP3 under the control of EF1α promoter and expressing the reporter protein ΔLNGFR under the control of PGK promoter, and its mock counterpart LNGFRp-e, and LNGFRe-pFOXP3 expressing FOXP3 under the control of PGK promoter and expressing the reporter protein ΔLNGFR under the control of the EFS promoter, and its mock counterpart LNGFRe-p. Two bicistronic vectors under the control of EF1 promoter, both based on a self-cleaving T2A sequence: eLNGFR.t2a.FOXP3 and its mock counterpart eLNGFR.t2a and eFOXP3.t2a.LNGFR, and its mock counterpart e.t2a.LNGFR. (B) Quantification of the titers of vector used for transduction. (C) Representative flow cytometry dot plots showing the expression of hFOXP3 and ΔLNGFR on WT murine CD4+ T cells 5 days after transduction with all the constructs expressing FOXP3. The correlation between FOXP3 expression and ΔLNGFR expression was quantified by calculating the Spearman correlation coefficient (r2 = 0.51, 0.54, 0.66, and 0.61 for the LNGFRp-eFOXP3, LNGFRe-pFOXP3, eLNGFR.t2a.FOXP3, and eFOXP3.t2a.LNGFR vectors, respectively). (D) Transduction efficacy quantified as the percentage of ΔLNGFR on day 5 after transduction in WT and scurfy CD4+ T cells. Transduction efficiency was significantly higher with the LNGFRp-eFOXP3 vector than with the LNGFR.t2a.FOXP3 vector for both WT CD4+ T cells and scurfy CD4+ T cells (P = .002 and .007, respectively, in a Mann-Whitney test). In contrast, transduction efficacy with the mock vectors was higher with the T2A construct (P = .02 and .04 in WT and scurfy CD4+ T cells, respectively). n = 3 independent experiments for WT CD4+ T cells and n = 2 independent experiments for scurfy CD4+ T cells. (E) The geometric MFI for FOXP3 expression was quantified on day 5 posttransduction (gated on CD4+ ΔLNGFR+) in WT cells or scurfy cells. The hFOXP3 MFI in WT CD4 T cells was similar with LNGFRp-eFOXP3 and LNGFR.t2a.FOXP3 vectors. The MFI was significantly higher in scurfy CD4 T cells transduced with the LNGFRp-eFOXP3 vector than in the same cell type transduced with LNGFR.t2a.FOXP3 (P = .04 in a Mann-Whitney test). n = 3 independent experiments for WT CD4+ T cells and n = 2 independent experiments for scurfy CD4+ cells. ψ, psi packaging element; *P < .05; **P < .01; APC, allophycocyanine; CMV, cytomegalovirus; FITC, fluorescein isothiocyanate; LTR, long terminal repeat; ns, not significant; PolyA, polyadenylation sequence; SIN, self-inactivating; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element.

The efficiency of FOXP3 expression by lentiviral vectors. (A) Vector maps showing the design of the 4 vectors and their mock counterparts within the pCCL backbone. Two bidirectional vectors: LNGFRp-eFOXP3 expressing FOXP3 under the control of EF1α promoter and expressing the reporter protein ΔLNGFR under the control of PGK promoter, and its mock counterpart LNGFRp-e, and LNGFRe-pFOXP3 expressing FOXP3 under the control of PGK promoter and expressing the reporter protein ΔLNGFR under the control of the EFS promoter, and its mock counterpart LNGFRe-p. Two bicistronic vectors under the control of EF1 promoter, both based on a self-cleaving T2A sequence: eLNGFR.t2a.FOXP3 and its mock counterpart eLNGFR.t2a and eFOXP3.t2a.LNGFR, and its mock counterpart e.t2a.LNGFR. (B) Quantification of the titers of vector used for transduction. (C) Representative flow cytometry dot plots showing the expression of hFOXP3 and ΔLNGFR on WT murine CD4+ T cells 5 days after transduction with all the constructs expressing FOXP3. The correlation between FOXP3 expression and ΔLNGFR expression was quantified by calculating the Spearman correlation coefficient (r2 = 0.51, 0.54, 0.66, and 0.61 for the LNGFRp-eFOXP3, LNGFRe-pFOXP3, eLNGFR.t2a.FOXP3, and eFOXP3.t2a.LNGFR vectors, respectively). (D) Transduction efficacy quantified as the percentage of ΔLNGFR on day 5 after transduction in WT and scurfy CD4+ T cells. Transduction efficiency was significantly higher with the LNGFRp-eFOXP3 vector than with the LNGFR.t2a.FOXP3 vector for both WT CD4+ T cells and scurfy CD4+ T cells (P = .002 and .007, respectively, in a Mann-Whitney test). In contrast, transduction efficacy with the mock vectors was higher with the T2A construct (P = .02 and .04 in WT and scurfy CD4+ T cells, respectively). n = 3 independent experiments for WT CD4+ T cells and n = 2 independent experiments for scurfy CD4+ T cells. (E) The geometric MFI for FOXP3 expression was quantified on day 5 posttransduction (gated on CD4+ ΔLNGFR+) in WT cells or scurfy cells. The hFOXP3 MFI in WT CD4 T cells was similar with LNGFRp-eFOXP3 and LNGFR.t2a.FOXP3 vectors. The MFI was significantly higher in scurfy CD4 T cells transduced with the LNGFRp-eFOXP3 vector than in the same cell type transduced with LNGFR.t2a.FOXP3 (P = .04 in a Mann-Whitney test). n = 3 independent experiments for WT CD4+ T cells and n = 2 independent experiments for scurfy CD4+ cells. ψ, psi packaging element; *P < .05; **P < .01; APC, allophycocyanine; CMV, cytomegalovirus; FITC, fluorescein isothiocyanate; LTR, long terminal repeat; ns, not significant; PolyA, polyadenylation sequence; SIN, self-inactivating; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element.

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