1. Volume 16, Issue 12, Pages 2011-2021 (December 2008)
Efficient Modulation of T-cell Response by Dual-mode, Single-carrier Delivery of Cytokine-targeted siRNA and DNA Vaccine to Antigen-presenting Cells 
Ankur Singh, Hui Nie, Bilal Ghosn, Hong Qin, Larry W Kwak, Krishnendu Roy 
Molecular Therapy 
Volume 16, Issue 12, Pages (December 2008)
DOI: /mt
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
In vitro characterization of interleukin 10 (IL-10) small interfering RNA (siRNA) encapsulated PEI–PLGA microparticles. (a) Bioactivity studies of siRNA: glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene knockdown in HEK293T cells using microparticle extracted Silencer GAPDH siRNA complexed with siPORT Amine. (b) In vitro IL-10 siRNA release from poly(lactide-co-glycolide) (PLGA) microparticles with varying polymer molecular weights. PEI, polyethyleneimine.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Simultaneous in vitro DNA transfection and gene silencing with plasmid DNA (pDNA) loaded interleukin 10 (IL-10) small interfering RNA (siRNA) encapsulated PEI–PLGA microparticles. Fluorescence images representing pEGFP-N3 transfection in (a–e) primary bone marrow–derived antigen-presenting cells (APCs) using pEGFP-N3-EXGEN500 [commercially available linear polyethyleneimine (PEI)-based transfection agent], pEGFP-N3-IL-10 siRNA–PEI–PLGA microparticles and IL-10 siRNA–PEI–PLGA microparticles. (f) IL-10 gene silencing in bone marrow–derived primary APCs after 48 hours, 5 days, and 15 days post transfection with pEGFP-N3-IL-10 siRNA–PEI–PLGA microparticles (g) Firefly Luciferase transfection in primary APCs. All samples were evaluated for Luciferase gene expression levels using real time RT-PCR and normalized to β-actin housekeeping gene, (h) IL-10 gene silencing in bone marrow–derived primary APCs after 48 hours, 5 days, and 15 days post transfection with pgWizLuciferase IL-10 siRNA–PEI–PLGA microparticles (quantified using real time RT-PCR), (i) IL-10 cytokine downregulation in bone marrow–derived primary APCs after 48 hours [quantified using enzyme-linked immunosorbent assay (ELISA)]. Primary APCs (1 × 106) were harvested and exposed to various formulations; supernatants were collected from culture medium and analyzed by ELISA for IL-10 production. Negative controls: untreated cells and cells treated with microparticles encapsulating scrambled siRNA (Silencer Negative Control #1 siRNA; Ambion). pDNA-loaded microparticles were also considered as negative control in ELISA studies. Positive control: IL-10 siRNA complexed to siPORT Amine. *P < 0.05 compared to negative controls. Bar = 20 µm. PLGA, poly(lactide-co-glycolide).
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Upregulation of cell surface markers in primary antigen-presenting cells (APCs). 2 × 105 bone marrow–derived APCs isolated from Balb/c mice were incubated with interleukin 10 (IL-10) siRNA–pDNA–PEI–PLGA, pDNA–PEI–PLGA, IL-10 siRNA–PEI–PLGA, and PEI–PLGA microparticles. DNA used in the study was pgWizBetaGal. Lipopolysaccharide (LPS) was used as positive control and incubation was followed for 48 hours. (a) Representative flow cytometry histogram of particle-treated cells overlaid with respect to untreated controls. Numbers represent gated cell percentages. (b) Bar graph representing average percentage (n = 3) of APCs expressing CD40, 41BBL, and CD 86 costimulatory molecules when transfected with dual-delivery system or single nucleic acid delivery system. (c) Bar graph showing average percentage of APCs expressing CD40 and CD86 costimulatory molecules when transfected with interleukin 10 (IL-10) specific or scrambled small interfering RNA (siRNA) (Silencer Negative Control #1 siRNA) carrying PEI–PLGA microparticles (n = 3). *P < 0.05 compared to IL-10 siRNA PEI–PLGA and PEI–PLGA; †P < 0.05 compared to pDNA–PEI–PLGA; ‡P < 0.05 compared to scrambled (mock) siRNA–PEI–PLGA and PEI–PLGA; #P < 0.05 compared to scrambled siRNA–PEI–PLGA. pDNA, plasmid DNA; PEI, polyethyleneimine; PLGA, poly(lactide-co-glycolide).
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Allogeneic T-cell proliferation in presence of activated primary antigen-presenting cells (APCs). 1 × 106 splenocytes isolated from C57BL/6 mice were incubated with activated bone marrow–derived allogeneic APCs at APC:T ratio of 1:4 and incubated for 3 days. Horizontal axis represents various treatment modalities for APCs (control or microparticle treated). Data are presented as mean ± SD of n = 3. *P < 0.05 compared to all other groups; #P < 0.05 compared to scrambled siRNA–PEI–PLGA, PEI–PLGA, and untreated cells. IL-10, interleukin 10; LPS, lipopolysaccharide; pDNA, plasmid DNA; PEI, polyethyleneimine; PLGA, poly(lactide-co-glycolide); siRNA, small interfering RNA.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
In vivo studies in a murine model of gWizHBsAg immunization. (a) Time line of immunization and T-cell response analysis, (b) dot plot exhibiting distribution of interferon γ (IFN-γ) or interleukin 4 (IL-4) expressing splenocytes. CD4+ cells (upper population) were gated and analyzed for IFN-γ and IL-4 production levels. (c) Representative quadrant analysis of gated CD4+ cells (pooled samples from n = 4 mice) for IFN-γ and IL-4 expression at 6 and 9 weeks. Splenocytes from treated animals were exposed to HBsAg peptide for cytokine production. Media pulsed splenocytes were used as negative controls for each group. Percentages indicate cells double positive for CD4 and cytokines. (d) Bar graph representing fold change in cells expressing IFN-γ for each group. For normalization, percentage values from media pulsed splenocytes were subtracted from HBsAg-pulsed splenocytes and expressed as a ratio of phosphate-buffered saline (PBS)-treated animals i.e., sample (HBsAg pulsed – media pulsed) divided by PBS (HBsAg pulsed – media pulsed). (e) Bar graph representing fold change in cells expressing IL-4 for each group. For normalization, expression values from media pulsed splenocytes were subtracted from HBsAg-pulsed splenocytes and expressed as a ratio of PBS-treated animals. *These groups did not show detectable cytokine production. PEI, polyethyleneimine; PLGA, poly(lactide-co-glycolide); siRNA, small interfering RNA.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
T cell–mediated cytotoxicity evaluation using Granzyme B assay. P815 target cells were pulsed with HBsAg or lymphocytic choriomeningitis virus (LCMV) (control) peptides and incubated with splenocytes (pooled samples from n = 4 mice). Granzyme B activity in P815 cells was determined using flow cytometry. (a) Dot plot exhibiting distribution of Granzyme B–expressing P815 target cells at 6 and 9 weeks time points. P815 cells were gated as shown. (b) Bar graph showing fold change in Granzyme B–expressing P815 cells exposed to splenocytes obtained from control or microparticle-treated mice groups. For normalization, percentage values from LCMV-pulsed cells were subtracted from HBsAg-pulsed cells and expressed as a ratio of phosphate-buffered saline (PBS)-treated animals. (c) Representative quadrant analysis of gated P815 cells for Granzyme B activity at 6 and 9 weeks time points. LCMV peptide–pulsed P815 cells were used as negative controls for each group. (d) Bar graph showing fold change in Granzyme B expressing P815 cells in the repeat in vivo studies with scrambled small interfering RNA (siRNA) control. P815 cells were exposed to splenocytes obtained from control or microparticle-treated mice groups. For normalization, percentage values from LCMV-pulsed cells were subtracted from HBsAg-pulsed cells and expressed as a ratio of PBS-treated animals. *These groups did not show detectable Granzyme B activity. IL-10, interleukin 10; PEI, polyethyleneimine; PLGA, poly(lactide-co-glycolide).
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions