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

Abstract

Silencing gene expression by RNAi is a powerful method for exploring gene function and validating drug targets and potentially for therapy. Lymphocytes and other primary blood cells are resistant to lipid-based transfection in vitro and are difficult to target in vivo. We show here that antibody-protamine fusion proteins targeting the human integrin lymphocyte function-associated antigen-1 (LFA-1) efficiently deliver siRNAs and specifically induce silencing in primary lymphocytes, monocytes, and dendritic cells. Moreover, a fusion protein constructed from an antibody that preferentially recognizes activation-dependent conformational changes in LFA-1 selectively targets activated leukocytes and can be used to suppress gene expression and cell proliferation only in activated lymphocytes. The siRNA-fusion protein complexes do not cause lymphocyte activation or induce IFN responses. K562 cells expressing latent WT or constitutively activated LFA-1 engrafted in the lungs of SCID mice are selectively targeted by intravenously injected fusion protein-siRNA complexes, demonstrating the potential in vivo applicability of LFA-1-directed siRNA delivery.

Fig. 1.

Selective targeting of siRNAs to PBMC expressing HA LFA-1 by AL-57-PF. PBMC were either unstimulated (1 mM MgCl₂, 1 mM CaCl₂) or stimulated with 5 mM MgCl₂, 1 mM EGTA, and 10 μg/ml CBRLFA-1/2 to activate LFA-1. (A) Activation-independent binding of TS1/22-PF and activation-dependent binding of AL-57-PF. (B) Selective delivery of Cy3-siRNA (1 nmol) to stimulated or unstimulated PBMC, measured 6 h after treatment. The LFA-1 antibody fusion proteins selectively delivered siRNAs to T lymphocytes (stained with CD3), B lymphocytes (CD19), monocytes (CD14), and dendritic cells (CD11c). (C) Silencing of Ku70 in PBMC. Ku70 expression was measured 3 d after treatment with Ku70-siRNA, delivered as indicated. (D) Silencing of CCR5 in T lymphocytes. Memory T lymphocytes were treated for 3 d in the presence or absence of LFA-1-activating antibody with 1 nmol of CCR5-siRNA, delivered as indicated. Expression of CCR5 mRNA relative to β-actin mRNA was measured by quantitative RT-PCR.

Fig. 2.

Selective silencing of Ku70 in mixed populations of K562 cells transfected to express LFA-1. CMTMR-labeled CBRLFA-1/2-activated cells, expressing HA LFA-1, were cocultured with unlabeled cells treated with an LFA-1 nonactivating antibody that express low-affinity LFA-1. Three days after treatment with 1 nmol of Ku70-siRNA delivered as indicated, the cocultures were analyzed for Ku70 silencing. AL57-PF-delivered siRNAs silence only the labeled activated cells (D), whereas TS-1/22-PF-delivered siRNAs silence Ku70 in both populations (C).

Fig. 3.

Persistent physiological stimulation of memory T cells activates sustained AL-57-PF binding and siRNA delivery. (A–C) Kinetics of affinity up-regulation of LFA-1 after activation of T cells. Cells stimulated for the indicated times with immobilized CXCL12 or anti-CD3 were analyzed for binding of Alexa-488-labeled fusion proteins. (D) Cy3-siRNA and Alexa-488-TS1/22-PF were taken up by unstimulated T cells, whereas uptake of Alexa- 488-AL-57-PF required T cell activation. (E) Activation-dependent silencing of Ku70 in T cells measured 3 d after treatment with 1 nmol of Ku70-siRNA delivered by scFv-PF. Mean fluorescence intensities (MFI) of representative histograms are shown.

Fig. 4.

Selective inhibition of proliferation by AL-57-PF-delivered cyclin D1-siRNA to activated T cells. Proliferation was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) incorporation 3 d after treatment with or without immobilized activating antibodies, combined with cyclin D1 or control siRNA complexed with scFv-PF fusion proteins, TS1/22 scFv, protamine, or medium. Silencing cyclin D1 using TS1/22-PF stopped proliferation of all T cells, whereas inhibition of proliferation using AL-57-PF required cell activation. ∗, P < 0.03; ∗∗, P < 0.01.

Fig. 5.

Specific in vivo siRNA delivery by anti-LFA-1 fusion proteins to K562 cells expressing human WT LFA-1 or human HA LFA-1. Four hours after injection of Cy3-siRNA complexed with AL-57- or TS1/22-PF, siRNA delivery to K562 cells in the lungs of SCID mice was examined by fluorescence microscopy. Anti-human CD45 labeled K562 cells. TS1/22-PF delivered siRNA equally well to cells expressing WT and HA-LFA-1. By contrast, AL-57-PF preferentially delivered to K562-HA LFA-1. Mouse lung cells did not take up the siRNA.