TLR9-Targeted SiRNA Delivery In Vivo

Abstract

The SiRNA strategy is a potent and versatile method for modulating expression of any gene in various species for investigational or therapeutic purposes. Clinical translation of SiRNA-based approaches proved challenging, mainly due to the difficulty of targeted SiRNA delivery into cells of interest and the immunogenic side effects of oligonucleotide reagents. However, the intrinsic sensitivity of immune cells to nucleic acids can be utilized for the delivery of SiRNAs designed for the purpose of cancer immunotherapy. We have demonstrated that synthetic ligands for the intracellular receptor TLR9 can serve as targeting moiety for cell-specific delivery of SiRNAs. Chemically synthesized CpG-SiRNA conjugates are quickly internalized by TLR9-positive cells in the absence of transfection reagents, inducing target gene silencing. The CpG-SiRNA strategy allows for effective targeting of TLR9-positive cells in vivo after local or systemic administration of these oligonucleotides into mice.

Keywords: Cancer; CpG; Leukemia; Myeloid cells; Oligonucleotides; SiRNA; TLR9.

Fig. 1

Quality control for the hybridized CpG-siRNA constructs. Mouse and human versions of siRNA(SS), CpG-siRNA(AS), and CpG-siRNA conjugate were loaded on the 7 M urea/15 % PAGE gel. After electrophoresis gel was stained using ethidium bromide to visualize oligonucleotides compared to a dsRNA marker

Fig. 2

Human immune and leukemic cells efficiently internalize CpG-siRNA conjugates in vitro. (a) Targeted delivery of CpG-STAT3 siRNA into different primary human immune cells in vitro. Human PBMCs were incubated with fluorescently labeled CpG(A)-STAT3 siRNA^Cy3 or unconjugated STAT3 siRNA^Cy3 at 500 nM concentration for 1 h without any transfection reagents. Percentages of Cy3⁺ monocytes (CD14⁺), plasmacytoid dendritic cells (BDCA2⁺), B cells (CD19⁺), and T cells (CD3⁺) were assessed by flow cytometry. (b) CpG-siRNA internalization by human MM (KMS.11) and AML (MV4-11, KG1a) cells. Cells were incubated with 500 nM Cy3-labeled CpG-STAT3 siRNA for 1 h. The percentage of Cy3⁺ leukemic cells was analyzed by flow cytometry

Fig. 2

Human immune and leukemic cells efficiently internalize CpG-siRNA conjugates in vitro. (a) Targeted delivery of CpG-STAT3 siRNA into different primary human immune cells in vitro. Human PBMCs were incubated with fluorescently labeled CpG(A)-STAT3 siRNA^Cy3 or unconjugated STAT3 siRNA^Cy3 at 500 nM concentration for 1 h without any transfection reagents. Percentages of Cy3⁺ monocytes (CD14⁺), plasmacytoid dendritic cells (BDCA2⁺), B cells (CD19⁺), and T cells (CD3⁺) were assessed by flow cytometry. (b) CpG-siRNA internalization by human MM (KMS.11) and AML (MV4-11, KG1a) cells. Cells were incubated with 500 nM Cy3-labeled CpG-STAT3 siRNA for 1 h. The percentage of Cy3⁺ leukemic cells was analyzed by flow cytometry

Fig. 3

Intracellular localization of CpG-siRNA after in vitro uptake. Representative images from time-lapse confocal microscopy of cultured primary bone-marrow derived macrophages incubated in the presence of 500 nM CpG-siRNA^Cy3 for 0.5 h

Fig. 4

CpG-Stat3 siRNA is efficiently internalized by AML cells in vivo. C57BL/6 mice bearing Cbfb-MYH11/Mpl+ AML were injected with a single dose of CpG-Stat3 siRNA^Cy3 (5 mg/kg). Uptake of fluorescently labeled siRNA was assessed using flow cytometry in AML cells isolated from spleen and bone marrow 3 or 18 h after injection

Fig. 5

Schematic of blood, bone, and soft tissue processing to assess organ and cellular biodistribution of CpG-siRNA conjugates