Polyethylenimine-based siRNA nanocomplexes reprogram tumor-associated dendritic cells via TLR5 to elicit therapeutic antitumor immunity

Abstract

The success of clinically relevant immunotherapies requires reversing tumor-induced immunosuppression.Here we demonstrated that linear polyethylenimine-based (PEI-based) nanoparticles encapsulating siRNA were preferentially and avidly engulfed by regulatory DCs expressing CD11c and programmed cell death 1-ligand 1 (PD-L1) at ovarian cancer locations in mice. PEI-siRNA uptake transformed these DCs from immunosuppressive cells to efficient antigen-presenting cells that activated tumor-reactive lymphocytes and exerted direct tumoricidal activity, both in vivo and in situ. PEI triggered robust and selective TLR5 activation in vitro and elicited the production of hallmark TLR5-inducible cytokines in WT mice, but not in Tlr5 -/-littermates. Thus, PEI is a TLR5 agonist that, to our knowledge, was not previously recognized. In addition, PEI-complexed nontargeting siRNA oligonucleotides stimulated TLR3 and TLR7. The nonspecific activation of multiple TLRs (specifically, TLR5 and TLR7) reversed the tolerogenic phenotype of human and mouse ovarian tumor-associated DCs. In ovarian carcinoma-bearing mice, this induced T cell-mediated tumor regression and prolonged survival in a manner dependent upon myeloid differentiation primary response gene 88 (MyD88; i.e., independent of TLR3). Furthermore, gene-specific siRNA-PEI nanocomplexes that silenced immunosuppressive molecules on mouse tumor-associated DCs elicited discernibly superior antitumor immunity and enhanced therapeutic effects compared with nontargeting siRNA-PEI nanocomplexes. Our results demonstrate that the intrinsic TLR5 and TLR7 stimulation of siRNA-PEI nanoparticles synergizes with the gene-specific silencing activity of siRNA to transform tumor-infiltrating regulatory DCs into DCs capable of promoting therapeutic antitumor immunity.

Enhancement of plasmid DNA immunogenicity with linear polyethylenimine

“…PEI is superior to other nonmicrobial transfection agents because of its ability to protect DNA from degradation [18], to escape intracellular endosomal lysis [19] and to efficiently deliver DNA into the nucleus of cells [20]. Formulation with PEI polymers has been employed in DNA-and siRNAbased immunotherapy against cancer [21,22] and in vaccination studies targeting different infectious agents [10,23,24].In the present study, we have evaluated the physicochemical characteristics of a series of PEI polymers and have assessed associations between particular characteristics of these polymers and their ability to facilitate in vivo DNA expression and enhanced adaptive immune responses. Moreover, we have examined plasmid DNA constructs formulated with these various PEI polymers for their ability to generate immune responses under conditions that maximize the immunogenicity of these PEI-DNA complexes.…”

Multifunctional nanoparticles for cancer immunotherapy

“…For example, linear PEI-based NPs encapsulating siRNA were used to silence PD-L1 expression on mouse tumor-associated DCs. 88 In addition, in this study linear PEI was identified as a novel TLR5 agonist and indicates that activation of TLR5 and TLR7 reversed the tolerogenic phenotype of human and mouse ovarian tumor-associated DCs. The results of this study demonstrated that transforming ovarian cancer-associated DCs in vivo from an immunosuppressive to an immunostimulatory and tumoricidal cell type is not only feasible using siRNA-PEI nanocomplexes, but also more effective against aggressive ovarian tumors than what previously reported synergistic effect of standard chemotherapies combined with DC depletion.…”

“…The results of this study demonstrated that transforming ovarian cancer-associated DCs in vivo from an immunosuppressive to an immunostimulatory and tumoricidal cell type is not only feasible using siRNA-PEI nanocomplexes, but also more effective against aggressive ovarian tumors than what previously reported synergistic effect of standard chemotherapies combined with DC depletion. 88 …”

Modulation of Cancer Progression by Tumor Microenvironmental Leukocyte-Expressed microRNAs

“…The caveat of this approach is that, as commented above, a myriad of phagocytic cells with enhanced endocytic pathways are present in the microenvironment of virtually all solid tumors. Overcoming endocytosis by these abundant leukocytes and reaching cancer cells represents a barrier that, at least in our hands, has proven impossible (Cubillos-Ruiz et al, 2009a, 2009bCubillos-Ruiz et al, 2010). Nevertheless, the myeloid leukocytes that spontaneously take up particulate materials are also optimal targets for miRNA mimeticsbased interventions.…”