siRNA Specific Delivery System for Targeting Dendritic Cells

Abstract

siRNA therapy offers immense potential for clinical application. Under physiological conditions, however, siRNA was demonstrated to have a short half-life. Additionally, it may also cause ubiquitous gene silencing as it does not possess a tissue-specific homing mechanism. Thus, the rate-limiting step in the emergence of siRNA as a potential therapeutic agent is the current lack of a safe and tissue- or cell-specific in vivo delivery system. Herein, we propose a novel, cell-specific method for the in vivo delivery of siRNA to dendritic cells (DCs) with the purpose of inducing immune modulation. CD40 siRNA was incorporated within the interior of 86 nm liposomes, which were decorated with surface-bound mAb NLDC-145 as a targeting mechanism. The siRNA encapsulation efficiency was determined to be approximately 7%. CD40 siRNA immunoliposomes (CD40 siILs) were able to specifically bind to DCs and silence CD40 expression in vitro. Furthermore, in vitro CD40-silenced DCs significantly inhibited the proliferation of alloreactive T cells in an MLR. Upon in vivo administration, siIL-encapsulated, Cy3-labeled siRNA exhibited moderate uptake by the liver at an early time point following administration with greater accumulation in the spleen at a later time point. In contrast, naked siRNA primarily accumulated in the kidney immediately after administration and circulated out in a short time period. To address in vivo gene silencing and immune modulation, mice were simultaneously immunized with KLH and subcutaneously injected with DC-specific CD40 siILs, siILs containing negative control siRNA, naked CD40 siRNA, or PBS. A second injection of CD40 siILs, or control treatments, followed 24 h later. Flow cytometry, reverse transcriptase PCR, and quantitative real-time PCR analysis of CD11c⁺ DCs from mice treated with CD40 siILs demonstrated reduced expression of CD40, in comparison with control groups. CD11c⁻ cells were also analyzed by flow cytometry, but no differences were observed between treatment groups. Furthermore, CD40 siIL-treated mice were found to have an increased proportion of Treg cells (CD4⁺CD25⁺ FoxP3⁺), and DCs cells from these mice were able to inhibit T cell proliferation in an antigen-specific recall response. In summary, CD40 siILs were shown to specifically target and deliver CD40 siRNA to DCs, significantly reducing CD40 expression and resulting in DC-mediated immune modulation as well as generation of Treg cells. These findings highlight the therapeutic potential for siRNA-based and DC-mediated immunotherapy in the clinic. To the best of our knowledge, this is the first study to use siILs for targeted delivery of siRNA to DCs and for immune modulation.