In vivo delivery of siRNA to immune cells by conjugation to a TLR9 agonist enhances antitumor immune responses

Abstract

Efficient delivery of siRNA to specific cell populations in vivo remains a formidable challenge to its successful therapeutic application. We describe a novel siRNA-based approach – synthetically linking siRNA to an oligonucleotide TLR9 agonist – that targets and silences genes in TLR9+ myeloid cells and B cells, both of which are key components of the tumor microenvironment. Because Stat3 in tumor-associated immune cells suppresses antitumor immune responses and hinders TLR9-induced immune stimulation, we tested CpG-Stat3siRNA conjugates for anti-tumor effects. When injected locally at the tumor site or systemically through an intravenous route, the CpG-Stat3siRNA conjugates access tumor-associated dendritic cells, macrophages and B cells, inhibit Stat3 expression, leading to activation of tumor-associated immune cells, and ultimately potent anti-tumor immune responses. Our findings demonstrate the potential of TLR agonist-siRNA conjugates for targeted gene silencing coupled with TLR stimulation and immune activation in the tumor microenvironment.

High doses of CpG oligodeoxynucleotides stimulate a tolerogenic TLR9–TRIF pathway

“…In contrast, higher dosages trigger TLR9-and IDO1-dependent tolerogenesis, which abrogates the stimulatory effects and relies on TRIF and the noncanonical NF-kB signalling pathway as well as on TGF-b production, which may further exploit IDO1 as a signalling protein in a feedforward loop involving the stable conditioning of pDCs towards a regulatory phenotype 12 . These data may have an impact on novel CpG-based therapeutic approaches in the context of antitumor and vaccination strategies, including siRNA delivery approaches targeting phagocytic cells through an siRNA conjugated to a TLR9 ligand 1 . These studies may also lead to innovative means of treating autoimmune and allergic inflammation.…”

“…TLR9, which recognizes unmethylated cytosine-phosphateguanosine (CpG) motifs, is a very promising target for therapeutic activation. Stimulation of TLR9 activates human plasmacytoid dendritic cells (pDCs) and B cells, and results in potent Th1-type immune reactivity and antitumor responses in mouse tumour models and in patients 1 . While CpG-rich oligodeoxynucleotides (CpG-ODNs) are potent immune activators in mice-accelerating and boosting antigen-specific immune responses by 5-500-fold 2 -their stimulatory effects are often less dramatic in humans, and this disparity between rodents and mammals has been attributed to differences in TLR9 expression in the different species 3 .…”

Special delivery: targeted therapy with small RNAs

“…One clever example takes advantage of native nucleic acid receptors on immune cells as a way to both activate innate immunity and induce gene silencing to improve the endogenous immune response to xenografted tumors. 44 Intratumoral or intravenous injection of siRNAs conjugated to a CpG oligonucleotide agonist of Toll-like receptor 9 targeted myeloid cells and B cells that express Toll-like receptor 9. These conjugated siRNAs simultaneously knock down Stat3 and activate Toll-like receptor 9 activation to induce antitumor immunity by reducing immunosuppressive regulatory T cells and enhancing tumor infiltration with antitumor killer T cells.…”

Therapeutic targeting in the silent era: advances in non-viral siRNA delivery

“…It has recently been shown that siRNA synthetically conjugated to a CpG oligonucleotide agonist of the TLR9, targeted and silenced the signal transducer and activator of transcription 3 (STAT3, an immune suppressor gene) in TLR9 (+) myeloid cells and B cells [113] . STAT3 has been reported to orchestrate the expression of immunosuppressive and angiogenic factors, contributing to a tumour environment characterized by a lack of tumour-specific cytotoxic T cells and an inhibition of T helper 1 (T H 1) cells [113] .…”

“…STAT3 has been reported to orchestrate the expression of immunosuppressive and angiogenic factors, contributing to a tumour environment characterized by a lack of tumour-specific cytotoxic T cells and an inhibition of T helper 1 (T H 1) cells [113] . Silencing of STAT3 using CpG-linked siRNA by either local delivery at the tumour site or intravenously injection in a mouse model led to activation of tumour-associated immune cells, triggering of antitumour immune responses and eventual inhibition of tumour growth [113] .…”