A Double-Switch Vector System Positively Regulates Transgene Expression by Endogenous microRNA Expression (miR-ON Vector)  Mario Amendola, Alice Giustacchini, Bernhard Gentner, Luigi Naldini  Molecular Therapy  Volume 21, Issue 5, Pages 934-946 (May 2013) DOI: 10.1038/mt.2013.12 Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Vector systems overview. (a) Schematic representation of the lentiviral vectors used in the study. Proviral forms are shown and arrows indicate orientation of transcription. From top: GFP reporter vector with the indicated number of tetO operator sequences inserted in different places; tet repressor encoding bidirectional LV; self-regulated bidirectional LV. (b) Schematic of system regulation in the indicated conditions. The repressor protein, either tTR or tTR-KRAB, constitutively binds the tetO operator and suppresses the expression of the reporter gene. When the repressor is not translated due to miR-mediated transcript degradation, or is unable to bind the tetO operator due to doxy-induced allosteric changes, GFP expression switches on. CMV, cytomegalovirus immediate early promoter; cPPT, central polypurine tract; ΔLTR, U3′ deleted long terminal repeat; GFP, green fluorescent protein; hPGK, human phosphoglycerate kinase promoter; LNGFR, low affinity nerve growth factor receptor; LTR, long terminal repeats; LV, lentiviral vectors; miRT, miR target sequence; pA, polyadenylation site; SFFV, spleen focus forming virus promoter; tetO, tetracycline operator; tTR, tetracycline repressor; tTR-KRAB, Kruppel-associated box; UTR, untranslated region; Wpre, woodchuck hepatitis virus posttranscriptional regulatory element. Molecular Therapy 2013 21, 934-946DOI: (10.1038/mt.2013.12) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 tTR-KRAB–dependent GFP repression. The indicated cell lines were transduced with the depicted LV.GFPtetO and, after 1 week, with the depicted LV.KRAB. Cells were kept with or without 1 µg/ml of doxy in the medium and 1 week later were FACS analyzed. Histograms show the distribution of GFP expression in the ΔLNGFR+ cells. GFP geometric MFI is indicated in each histogram. The black line (-, open area) indicates cells treated with doxy, the gray line (-, filled area) indicates cells without doxy. Four different LV.KRAB dilutions were tested (a) for each cell lines or (b) for each reporter vector with similar results. Representative plots are shown. FACS, fluorescence-activated cell sorter; ΔLTR, U3′ deleted long terminal repeat; GFP, green fluorescent protein; hPGK, human phoshoglycerate kinase promoter; LNGFR, low affinity nerve growth factor receptor; LTR, long terminal repeats; LV, lentiviral vectors; MFI, mean fluorescence intensity; pA, polyadenylation site; SFFV, spleen focus forming virus promoter; tetO, tetracycline operator; tTR-KRAB, Kruppel-associated box; Wpre, woodchuck hepatitis virus posttranscriptional regulatory element. Molecular Therapy 2013 21, 934-946DOI: (10.1038/mt.2013.12) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 tTR-dependent GFP repression. 293T cells were transduced with the depicted LV.GFPtetO and, after 1 week, with the tTR or tTR-KRAB encoding LVs. Cells were kept with or without 1 µg/ml of doxy in the medium and 1 week later were FACS analyzed. (a) Histograms show the distribution of GFP expression in the ΔLNGFR+ cells. GFP MFI is indicated in each histogram. The black line (-, open area) indicates cells treated with doxy, the gray line (-, filled area) indicates cells without doxy. (b) Left dot plot shows the gate strategy used to select cells to analyze. Right dot plots show the distribution of GFP and OFP expression in the ΔLNGFR+ cells with or without doxy, as indicated. Percentage of OFP+ cells and OFP MFI of the whole population is indicated in each dot plot. Four different LV.KRAB or LV.tTR dilutions were tested for each reporter vector with similar results. CMV, cytomegalovirus immediate early promoter; FACS, fluorescence-activated cell sorter; GFP, green fluorescent protein; hPGK, human phoshoglycerate kinase promoter; LNGFR, low affinity nerve growth factor receptor; LV, lentiviral vectors; MFI, mean fluorescence intensity; OFP, orange fluorescent protein; pA, polyadenylation site; tetO, tetracycline operator; tTR, tetracycline repressor; tTR-KRAB, Kruppel-associated box; Wpre, woodchuck hepatitis virus posttranscriptional regulatory element. Molecular Therapy 2013 21, 934-946DOI: (10.1038/mt.2013.12) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 miR regulation of LV.KRAB. The indicated hematopoietic and endothelial cell lines were transduced with the LV.GFPtetO and, after 1 week, with (a,b) the tTR-KRAB or (c) the tTR encoding LVs bearing the indicated miRT. Cells were kept with or without 1 µg/ml of doxy in the medium and 1 week later were FACS analyzed. Histograms show the distribution of GFP expression in the ΔLNGFR+ cells. GFP MFI is indicated in each histogram. The black line (-, open area) indicates cells treated with doxy, the gray line (-, filled area) indicates cells without doxy. Bar plots show the mean %GFP activation ± SEM relative to cells treated with doxy, calculated from the frequency of GFP+ cells. Note that the low frequency of GFP+ cells computed in the scrT sample can be well distinguished from the true positive cells in the miRT samples because of the much lower GFP MFI. P value of the Mann–Whitney test is indicated for each bar plot. (a,b) Three to (c) nine LV.miRT dilutions were tested for each cell line with similar results. CMV, cytomegalovirus immediate early promoter; FACS, fluorescence-activated cell sorter; GFP, green fluorescent protein; hPGK, human phoshoglycerate kinase promoter; LNGFR, low affinity nerve growth factor receptor; LV, lentiviral vectors; MFI, mean fluorescence intensity; miRT, miR target sequence; pA, polyadenylation site; SFFV, spleen focus forming virus promoter; tetO, tetracycline operator; tTR, tetracycline repressor; tTR-KRAB, Kruppel-associated box; Wpre, woodchuck hepatitis virus posttranscriptional regulatory element. Molecular Therapy 2013 21, 934-946DOI: (10.1038/mt.2013.12) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Monitoring miR expression in primary hematopoietic cells. Human cord blood cells were transduced with the LV.tTR-KRAB bearing the indicated miRNA target sequences and, 24 hours later, with the LV.GFPtetO. Cells were kept with or without 1 µg/ml of doxy in the medium and were FACS analyzed. (a) Histograms show the distribution of GFP expression in the bulk population. The MFI of GFP+ cells is indicated in each histogram. The black line (-, open area) indicates cells treated with doxy, the gray line (-, filled area) indicates cells without doxy. (b) Bar plot shows the %GFP activation relative to cells treated with doxy in the different subpopulations identified according to the gating strategy shown in the dot plot. One of the two experiments performed with similar results is shown. FACS, fluorescence-activated cell sorter; ΔLTR, U3′ deleted long terminal repeat; GFP, green fluorescent protein; hPGK, human phoshoglycerate kinase promoter; LNGFR, low affinity nerve growth factor receptor; LV, lentiviral vectors; MFI, mean fluorescence intensity; miRT, miR target sequence; pA, polyadenylation site; SFFV, spleen focus forming virus promoter; tetO, tetracycline operator; tTR-KRAB, Kruppel-associated box; Wpre, woodchuck hepatitis virus posttranscriptional regulatory element. Molecular Therapy 2013 21, 934-946DOI: (10.1038/mt.2013.12) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 In vivo application of miR-ON Technology. Human cord blood cells were transduced with the LV.GFPtetO, and 48 hours later, with the LV.tTR-KRAB bearing the miRNA target sequences (4 × 142T or 8 × 126T). After 6 days, double-transduced cells were FACS sorted for GFP and NGFR expression (see Supplementary Figure S3) and transplanted into NSG mice. Eighteen weeks later, bone marrow and spleen were collected and analyzed by FACS. (a) The FACS plots show the identification of human hematopoietic cells in the bone marrow of the mice (hCD45+) and the gating strategy to distinguish among different HSPC subpopulations according to CD34, CD38, CD45RA, and CD90 expression. (b) FACS plots show the GFP expression level within the different subpopulation from the bone marrow of the mice transplanted with indicated vector transduced cells. (c) Bar plot shows the mean %GFP+ cells in the different subpopulations of bone marrow or spleen identified according to the gating strategy shown in the dot plot. FACS, fluorescence-activated cell sorter; GFP, green fluorescent protein; HSPC, hematopoietic stem and progenitor cells; LV, lentiviral vectors; NGFR, nerve growth factor receptor; NSG, NOD/Lt-scid/IL2Rγnull; tTR-KRAB, Kruppel-associated box. Molecular Therapy 2013 21, 934-946DOI: (10.1038/mt.2013.12) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Self-regulated LV for miR-ON system. The indicated hematopoietic and endothelial cell lines were transduced with the tTR-KRAB self-regulated LVs bearing the indicated miR target sequences. Cells were kept with or without 1 µg/ml of doxy in the medium and 1 week later were FACS analyzed. Histograms show the distribution of GFP expression in cell population. Legend as in Figure 4. Four or five LV.miRT dilutions were tested for each cell line with similar results. ANOVA, analysis of variance; FACS, fluorescence-activated cell sorter; ΔLTR, U3′ deleted long terminal repeat; GFP, green fluorescent protein; LV, lentiviral vectors; MFI, mean fluorescence intensity; miRT, miR target sequence; pA, polyadenylation site; SFFV, spleen focus forming virus promoter; tetO, tetracycline operator; tTR-KRAB, Kruppel-associated box; Wpre, woodchuck hepatitis virus posttranscriptional regulatory element. Molecular Therapy 2013 21, 934-946DOI: (10.1038/mt.2013.12) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions