Selective gene silencing in activated leukocytes by targeting siRNAs to the integrin lymphocyte function-associated antigen-1

Peer et al. 10.1073/pnas.0608491104.

Supporting Information

Files in this Data Supplement:

SI Figure 6
SI Figure 7
SI Figure 8
SI Figure 9
SI Table 1
SI Figure 10
SI Methods

SI Figure 6

Fig. 6. siRNA delivery to PBMC by LFA-1 antibody-fusion proteins. Cells were unstimulated or stimulated with Mg/EGTA plus an activating mAb CBRLFA-1/2. (A) Stimulation with CBRLFA-1/2 did not affect LFA-1 expression on any subset. (B) Representative flow cytometry histograms show binding of Alexa 488-conjugated scFv-PF (20 mg/ml). Conformation-dependent AL-57-PF (solid lines) binds only to stimulated cells, while conformation-insensitive TS1/22-PF (dashed lines) binds to either unstimulated or stimulated cells and the control ErbB2 fusion protein ML39-PF (dotted lines) binds to neither.

SI Figure 7

Fig. 7. siRNA-mediated silencing of Ku70 in PBMC (A) or CD4 in CD4⁺ lymphocytes (B) Cells that were either unstimulated or stimulated with Mg/EGTA plus CBRLFA-1/2, were treated for 3 d with 1 nmol Ku70-siRNA or CD4-siRNA complexed with the indicated delivery reagents, and expression of Ku70 or CD4 was measured by flow cytometry. Data are mean ± SD of three independent experiments, normalized to expression of mock-treated cells.

SI Figure 8

Fig. 8. Additional controls to Fig. 2 confirm the specificity of Ku70-siRNA delivered with LFA-1 antibody fusion proteins in heterogeneous populations. CMTMR-labeled, CBRLFA-1/2-activated cells that express high affinity LFA-1 were cocultured with unlabeled, TS2/4-unactivated cells that express low-affinity LFA-1. Three days after treatment with 1 nmol Ku70-siRNA or luciferase-siRNA delivered as indicated, the cocultures were analyzed for Ku70 expression.

SI Figure 9

Fig. 9. Knockdown of cyclin D1 in T cells. IL-15-cultured T cells were treated for 60 h with 1 nmol siRNA mixed with protamine, TS1/22 scFv (TS1/22), or antibody-protamine fusion proteins (ML39-PF, TS1/22-PF, or AL-57-PF) in the presence of immobilized antibodies: 5 mg/ml anti-CD3 (CD3); a combination of 5 mg/ml anti-CD3 and 5 mg/ml anti-CD28 (CD3/CD28); or 5 mg/ml isotype control IgG (MOCK). Cells were fixed and permeabilized, and stained with Cyclin-D1-FITC mAb (clone DCS-6, Santa Cruz Biotechnology). Data are mean ± SD of three independent experiments and shown as a percentage of the mean fluorescent intensity of cyclin D1 in untreated cells. *P < 0.05, **P < 0.01. Silencing correlated with the specificity of the antibody and correlated with suppression of cellular proliferation shown in Fig. 4.

SI Figure 10

Fig. 10. Anti-LFA-1 scFv fusion protein-siRNA complexes do not activate lymphocytes or induce IFN responses in PBMC. (A and B) Cell surface expression of the activation markers CD69 and CD25 was measured by flow cytometry 2 d following treatment of PBMC with 1 nmol luciferase-siRNA complexed with indicated scFv-PF (dashed lines), siRNA alone (thick lines), or PHA (thin lines). (C) Expression of IFN responsive genes relative to b-actin analyzed by quantitative RT-PCR in CBRLFA-1/2-activated PBMC treated with luciferase-siRNA delivered as indicated. Poly (I:C) and LPS were used as positive controls to induce IFN responses. The siRNA complexes did not induce either cellular activation or an IFN response.

Table 1. In vivo siRNA delivery to K562 cells expressing LFA-1 in SCID mice

Cells	Cy3⁺ K562 cells/Total K562 cells, %
	Fusion protein	TS1/22-PF	AL-57-PF	ML39-PF
K562-WT LFA-1*		88.5 ± 7.0 (9)	23.3 ± 3.2 (9)	4.7 ± 2.2 (9)
K562-HA LFA-1**		87.4 ± 7.4 (6)	88.1 ± 6.8 (6)	4.2 ± 1.5 (6)

In vivo Cy3-siRNA delivery to K562 cells engrafted in the lungs of SCID mice was quantified by flow cytometry analysis of single-cell suspensions of lung tissues. K562 cells were identified by staining with FITC-human CD45 mAb. The number of mice used is shown in parentheses. Data are mean ± SD of three (*) and two (**) independent experiments.

SI Methods

Construction and expression of scFv and scFv-protamine fusion proteins. mRNA isolated from the TS1/22 hybridoma (provided by Timothy A. Springer, CBR Institute for Biomedical Research and Harvard Medical School) (1) was converted into cDNA. A plasmid containing AL-57 cDNA was previously described (2). Using overlapping PCR, the heavy and light chain variable domains of TS1/22 and AL-57 were amplified and engineered into an scFv plasmid with a (G₄S)₄ linker (3). The scFv cDNAs were subcloned into pET-26b (Novagen) that encodes for a C-terminal His-6 tail. To generate the scFv-protamine fragment fusion protein (scFv-PF) cDNA, overlapping PCR was used to fuse the scFv cDNA in frame to the N terminus of the cDNA fragment encoding Arg-8 to Ser-29 of human protamine, which was then subcloned into pET-26b. All constructs were verified by DNA sequencing. scFv and scFv-PF proteins were expressed in BL21(DE3) (Novagen), and purified from the periplasm by Ni-NTA affinity chromatography followed by ion-exchange chromatography with mono Q HR5/5 (Pharmacia) for scFv and mono S HR5/5 (Pharmacia) for scFv-PF. The pooled fractions were dialyzed against PBS and then PBS with 5% glycerol and stored at -80°C. The control scFv-PF fusion protein that recognizes human ErbB2 (ML39-PF) was previously described (4, 5).

PBMC and memory T lymphocytes. CD4 T cells were isolated from normal donor PBMC by selection with human CD4 immunomagnetic beads (Miltenyi Biotec). Memory T cells were prepared by culturing PBMC in RPMI 1640 medium containing 10% FCS for 3 d in the presence of 4 mg/ml phytohemagglutinin (PHA), followed by treatment with IL-15 (10 ng/ml) for 3 d.

Preparation of siRNAs. siRNAs from Dharmacon were deprotected and annealed according to the manufacturer's instructions. Four Ku70-siRNAs were used in an equimolar ratio as previously reported (6). CCR5- and CD4-siRNAs were previously reported (7, 8). Cyclin-D1-siRNAs (sc-29286) were from Santa Cruz Biotechnology (Santa Cruz, CA). The sense and anti-sense of siRNAs were: Cy3-luciferase, 5'-Cy3-CGUACGCGGAAUACUUCGAdTdT-3'(sense), 5'-UCGAAGUAUUCCGCGUACGdTdT-3' (antisense); luciferase, 5'-CGUACGCGGAAUACUUCGAdTdT-3'(sense), 5'-UCGAAGUAUUCCGCGUACGdTdT-3' (antisense).

siRNA transfection. siRNA transfection with PEI (ExGene 500, Fermentas Life Science), Oligofectamine (Invitrogen), and Fugene 6 (Roche) was preformed according to manufacturer's instructions.

Quantitative RT- PCR. Total RNA (1 mg) isolated with TRIzol (Invitrogen Life Technologies) was reverse-transcribed by using Superscript III (Invitrogen) and random hexamers, according to the manufacturer's protocol. Real-time quantitative PCR was performed on 1 ml of cDNA or a comparable amount of RNA with no reverse transcriptase, using Platinum Taq Polymerase (Invitrogen) and a Bio-Rad iCycler. SYBR green (Molecular Probes) was used to detect PCR products. All reactions were done in a 25-ml reaction volume in triplicate. The following primers were used:

CCR5 (forward), 5'-TGTTTGCGTCTCTCCCAGGAATCA-3'

CCR5 (reverse), 5'-AGCCCTGTGCCTCTTCTTCTCATT-3'

b-actin (forward), 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3'

b-actin (reverse), 5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3'

STAT1 (forward), 5'-CGGTTGAACCCTACACGAAG-3'

STAT1 (reverse), 5'-ACTTTCCAAAGGCATGGTC-3'

OAS1 (forward), 5'-GCAGAAAGAGGGCGAGTTC-3'

OAS1 (reverse), 5'-TACTGAGGTGGCAGCTTCC-3'

IFN b (forward), 5'-CCTGTTGTGCTTCTCCAC-3'

IFN b (reverse), 5'-ATGTCAAAGTTCATCCTGTC-3'

PCR parameters consisted of 5 min of Taq activation at 95°C, followed by 40 cycles of PCR at 95°C ´ 20 sec, 60°C ´ 30 sec, and 69°C ´ 20 sec. Standard curves were generated and the relative amount of target gene mRNA was normalized to b-actin mRNA. Specificity was verified by melt curve analysis and agarose gel electrophoresis.

Lymphocyte proliferation. Lymphocyte proliferation was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay as described (9).

Flow cytometry. Flow cytometry of cell surface antigens was performed as described (5). The following mAbs were used: FITC- or PE-conjugated mAbs to CD3, CD4, CD19, CD14, CD11c (BD Bioscience); APC-conjugated mAbs to CCR5 (BD Bioscience); FITC- or PE-conjugated mAbs to CD45 (Immunotech); FITC-conjugated anti-6His tag (Zymed); mAb to protamine (Santa Cruz Biotechnology); FITC and Cy3-conjugated anti-goat and anti-human Ig secondary antibodies (Zymed). mAbs to integrin aL (TS2/4, TS1/22) and b2 (TS1/18) subunits were gifts from Timothy A. Springer and were labeled with Alexa 488 using an Alexa dye kit (Invitrogen).

For measuring intracellular expression of cyclin D1, cells were fixed and permeabilized with the Fix-and-Perm kit (Caltag Laboratories, Burlingame, CA), stained with 1 mg/ml goat anti-human cyclin D1 (Santa Cruz Biotechnology) on ice for 30 min, and counterstained with FITC-conjugated rabbit anti-goat IgG (Zymed). Detection of Ku70 expression was as described (6).

Data were acquired and analyzed on FACScan or FACScalibur with CellQuest software (Becton Dickinson, Franklin Lakes, NJ).

Image acquisition and processing. Confocal imaging was performed using a Bio-Rad Radiance 2000 Laser-scanning confocal system (Hercules, CA) with an Olympus BX50BWI microscope using an Olympus 100X LUMPlanFL 1.0 water-dipping objective. Image acquisition was performed using Laserscan 2000 software and image processing was performed with Openlab 3.1.5 software (Improvision, Lexington, MA).

Immunohistochemistry. Frozen sections (5 mm thick) were air-dried, fixed in precooled acetone for 10 min, washed three times with PBS, and blocked with 10% FCS/PBS for 10 min. Sections were then incubated with FITC-labeled anti-human CD45 mAb (Immunotech) in 10% FCS/PBS overnight at 4°C in the dark. After washing three times with PBS, sections were incubated with 400 nM DAPI in PBS at room temperature for 10 min. Slides were washed three times with PBS, air-dried for 10 min, and mounted with VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA). Mounted slides were observed with an Axiovert 200M inverted microscope (Zeiss).

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