1. Volume 17, Issue 6, Pages 1039-1052 (June 2009)
Regulated and Multiple miRNA and siRNA Delivery Into Primary Cells by a Lentiviral Platform 
Mario Amendola, Laura Passerini, Ferdinando Pucci, Bernhard Gentner, Rosa Bacchetta, Luigi Naldini 
Molecular Therapy 
Volume 17, Issue 6, Pages (June 2009)
DOI: /mt
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Constitutive and self-regulated coexpression of miR and marker gene by LV. (a) Schematics of the LV used for expressing miR (left; LV.miR) and for reporting miR activity (right; LV.GFP.miRT). Proviral forms are shown, arrows indicate transcripts. For abbreviations see below. (b,c) HeLa cells were transduced with LV.GFP.223T and, after 1 week, with high and low MOI (right and left plots, respectively) of the depicted LV.223 or no-miR vectors (schematic of the expression cassette on the left). One week later cells were analyzed by FACS. The frequency of ΔLNGFR+ cells and their GFP (X) and ΔLNGFR (Y) MFI are indicated on top of each plot. (d) Representative western blot analysis of NFI-A expression in K562 cells either untreated or transduced with LV.223. Densitometric analysis of the NFI-A band is shown below, using GAPDH as normalizer, and the value are presented relative to no-miR LV transduced cells (64 ± 2%, mean ± SD). (e) GFP.223T HeLa, generated as above, were transduced with high (left) and low (right) MOI of the depicted LV.223 and no-miR vectors (LV.Ctrl) and analyzed as in b. Histograms show GFP expression of the ΔLNGFR+ cells. GFP MFI is indicated. Untreated cells—without GFP reporter—are shown for reference. (f) Schematic of the self-regulated LV.223. (g) GFP.223T HeLa were transduced with high (left) and low (right) MOI of the self-regulated LV.223 and a cognate no-miR vectors (LV.Ctrl), kept with or without 500 ng/ml doxycycline and analyzed as in b. (h) miR expression levels by RT-qPCR analysis in GFP.223T HeLa transduced with high MOI of the indicated self-regulated LV. All results shown are representative of at least two independent experiments performed on different batches of reporter cells and at ≥3 MOI (except for h, which was performed only for high MOI transduced cells) with similar results. CMV, immediate/early enhancer promoter of the human cytomegalovirus; cPPT, central polypurine tract; ctrl, control; GAPDH, glyceraldehyde 3 phosphate dehydrogenase; EF1α, human elongation factor 1 α promoter, 1st exon and intron; FACS, fluorescence activated cell sorting; GFP, green fluorescent protein cDNA; LV, lentiviral vector; MFI, mean fluorescence index; miR, microRNA; MOI, multiplicity of infection; NFI, nuclear factor I A; PGK, human phosphoglycerate kinase promoter; RRE, Rev responsive element; RT-qPCR, reverse transcription quantitative PCR; SA, splice acceptor site; SD, splice donor site; tetO7, tetracycline dependent promoter made by replacing the U3 sequence from position −418 to −36 with seven tandem repeats of tetracycline operators; tTA-2S, synthetic gene for the tetracycline dependent transactivator-2 cDNA; UT, untransduced cells; WPRE, woodchuck hepatitis virus post-transcription regulatory element; ΔLNGFR, truncated human low affinity nerve growth factor receptor cDNA; ΔU3, R and U5, HIV-1 LTR regions with −18 deletion relative to R in U3; ψ, encapsidation signal including the 5′ portion of the gag gene (GA); 223T, four tandem copies of a perfectly complementary target sequence for miR 223.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Multiple miR delivery. (a,c) HeLa cells were transduced with the indicated GFP.miRT reporter vectors and, after 1 week, transduced with high and low MOI (right and left histograms, respectively) of the depicted LV.miR and no-miR vectors (LV.Ctrl), and analyzed as in Figure 1. Histograms show GFP expression of the ΔLNGFR+ cells. GFP MFI is indicated. Untreated cells—without GFP reporter—are shown for reference. (b) miR expression levels by RT-qPCR analysis in GFP.miRT HeLa cells transduced with high MOI of the indicated LV. (d) Genomic DNA was extracted from GFP.miRT HeLa cells 10 days after transduction with high MOI of the indicated LV and PCR-analyzed for miR retention using an intronic forward primer and a 5′ ΔLNGFR reverse primer. Expected fragment lengths: no-miR LV = 631 bp, LV.223 = 943 bp, LV = 1,218 bp, LV = 1,217 bp. Results shown are from one of two independent experiments performed on different batches of reporter cells and at ≥3 MOI (except for b and d, which were performed only for high MOI transduced cells) with similar results. ctrl, control; EF1α, human elongation factor 1 α promoter, 1st exon and intron; GFP, green fluorescent protein cDNA; LV, lentiviral vector; MFI, mean fluorescence index; miR, microRNA; MOI, multiplicity of infection; RT-qPCR, reverse transcription quantitative PCR; SA, splice acceptor site; SD, splice donor site; WPRE, woodchuck hepatitis virus post-transcription regulatory element; ΔLNGFR, truncated human low affinity nerve growth factor receptor cDNA.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Heterologous miR expression. The pri-miR 223 upper stem–loop (sl) was replaced by that of other miR, as depicted on the left, and the chimeric miR and no-miR vectors (LV.Ctrl) were used to transduce at high and low MOI (left and right histograms, respectively) GFP.miRT HeLa cells as in Figure 1. Histograms show GFP expression of the ΔLNGFR+ cells. GFP MFI is indicated. Untreated cells—without GFP reporter—are shown for reference. Results shown are from one of two independent experiments performed on different batches of reporter cells and at ≥3 MOI with similar results. ctrl, control; GFP, green fluorescent protein cDNA; LV, lentiviral vector; MFI, mean fluorescence index; miR, microRNA; MOI, multiplicity of infection; WPRE, woodchuck hepatitis virus post-transcription regulatory element; ΔLNGFR, truncated human low affinity nerve growth factor receptor cDNA.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Expression and activity of mature miR 30 upon delivery within the pri-miR 223 or its own backbone in different cell types. The indicated cell lines were transduced with the GFP.30-3pT or GFP.30-5pT reporter LV and, after 1 week, with high and low MOI (left and right histograms, respectively) of the indicated LV.miR/chimeric miR and no-miR vectors (LV.Ctrl). Schematics of the parental and chimeric pri-miR are shown on top; sl, miR upper stem–loop. Cells were analyzed by FACS (left) and RT-qPCR (right), at 7 and 14 days after transduction, respectively, as in Figure 1. FACS histograms show GFP expression of the ΔLNGFR+ cells. GFP MFI is indicated. Untreated cells—without GFP reporter—are shown for reference. Results shown are from one of two independent experiments performed on different batches of reporter cells and at ≥3 MOI (except for RT-qPCR which was performed only for high MOI transduced cells) with similar results. Cell lines used: bEnd, murine brain endothelial; DH82, canine monocytic; JURKAT, human T lymphoblastoid; HaCat, human keratinocytic; HUH7, human hepatocytic; SHSY-5Y, human neuroblastoma; 293T, human kidney carcinoma. FACS, fluorescence activated cell sorting; GFP, green fluorescent protein cDNA; LV, lentiviral vector; MFI, mean fluorescence index; miR, microRNA; MOI, multiplicity of infection; RT-qPCR, reverse transcription quantitative PCR; ΔLNGFR, truncated human low affinity nerve growth factor receptor cDNA.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Northern blot analysis for miR 30 expression and processing by LVsl30 and LV.30. Cell lines were transduced with the indicated vectors at high MOI (same samples shown in Figure 4) and small RNA were extracted. Blot was probed for (a) miR 30-3p or (b) miR 30-5p. Left, the indicated number of copies of oligonucleotides corresponding to the pre-miR 30 (63 nucleotides) and mature miR 30-3p (22 nucleotides) were loaded on the gel as standards. Bottom, ethidium bromide shows similar RNA loading. a and b are the same filter. Arrow heads indicate the pre-miR band. Results shown are from one of two similar experiments performed. LV, lentiviral vector; miR, microRNA; MOI, multiplicity of infection; pri-miR, primary transcript.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
siRNA delivery by LV.amiR. (a) The pri-miR 223 upper stem was replaced by siRNA against the indicated gene of interest to generate an amiR, as shown in the schematic. (b) bEnd cells were transduced with LV.amiR against murine Tie2 (LV.siTie2) or luciferase (LV.siLuc) at high MOI and analyzed by FACS and RT-qPCR after 7 and 14 days, respectively. Left histogram shows the endogenous Tie2 expression of the ΔLNGFR+ cells. Tie2 MFI is indicated. Isotype control stained cells are shown for reference. Right bar histogram shows fold of Tie2 mRNA downregulation as calculated by RT-qPCR (error bars represent 95% of the confidence interval for each value). (c) U937 cells engineered to express murine Tie2 were transduced with LV.siTie2 and LV.siLuc vector at high MOI and analyzed as above. (d) U937-Tie2 cells were transduced with the indicated single and double amiR vector at low MOI and analyzed after 14 days as above. Tie2 MFI is indicated in the legend. (e) Reporter cell lines were generated by transducing HeLa cells with vectors encoding for GFP tagged with a fragment of the Tie2 cDNA (GFP.guide) or its complement (GFP.passenger). These cells were then transduced with LV.siTie2 and LV.siLuc vector at high MOI and analyzed as above. (f) U937 and HeLa cells, which naturally express or not miR223, were transduced with the depicted bicistronic LV carrying or not four copies of the miR 223 perfectly complementary target sequence. Both vectors efficiently express both marker genes in HeLa cells. Expression of both genes was suppressed in U937 cells from the miR.223T vector while expression of control vector was unaffected. (g) HeLa cells were engineered to express human FOXP3 by transduction with the depicted bicistronic LV and then transduced with LV.siFOXP3 and siLuc vector. Cells were analyzed by FACS after 10 days for FOXP3 and GFP expression. Histograms show FOXP3 and GFP expression of the ΔLNGFR+ cells. FOXP3 and GFP MFI are indicated. Untreated cells—without GFP reporter—are shown for reference. Results shown are representative of at least two independent experiments performed with similar results. ctrl, control; FACS, fluorescence activated cell sorting; FOXP3, Forkhead Transcription Factor P3; GFP, green fluorescent protein cDNA; IRES, internal ribosomal entry site; LV, lentiviral vector; MFI, mean fluorescence index; miR, microRNA; MOI, multiplicity of infection; RT-qPCR, reverse transcription quantitative PCR; UT, untransduced cells; WPRE, woodchuck hepatitis virus post-transcription regulatory element; ΔLNGFR, truncated human low affinity nerve growth factor receptor cDNA.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

8. Figure 7
Knockdown of FOXP3 expression and reversion of suppressive phenotype of human Treg cells by LV.siFOXP3. FACS sorted human CD4+CD25bright Treg cells were transduced with the indicated LV.siFOXP3 or LV.siLuc vector at MOI 20. After 14 days of culture cells were stained for FOXP3 expression. (a) Representative FACS density plots; percent of positive cells is indicated. UT, untransduced cells. (b) Percent FOXP3 positive cells (left) and MFI of FOXP3 staining relative to UT (right) of the indicated samples. Results are presented as mean ± SE. Significance assessed by Mann–Whitney test. (c) The ability of LV.siFOXP3 transduced Treg cells to suppress the proliferation of allogeneic CD4+ responder T cells was assessed at a 1:0.5 responder: Treg ratio by evaluating 3[H]-thymidine incorporation after stimulation with antigen presenting cells (APC) and anti-CD3 antibody and 3-day culture. In these conditions, LV.siFOXP3 Treg cells remained anergic. Representative experiment out of five performed with similar results. (d) Treg cells were activated by plate-bound anti-CD3 antibody before the proliferation assay. Results are presented as fold increase versus untrasduced Treg cells, tested in parallel. cpm, counts per minute; ctrl, control; FACS, fluorescence activated cell sorting; GFP, green fluorescent protein cDNA; LV, lentiviral vector; MFI, mean fluorescence index; miR, microRNA; MOI, multiplicity of infection; Treg, effector T cells; Teff, regulatory T cells; UT, untransduced cells.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions