a549 dual ko rig i  (InvivoGen)

Journal: bioRxiv

Article Title: Enhanced lung delivery of an immunostimulatory duplex RNA augments the antitumor activity by reshaping systemic cytokine pharmacodynamics

doi: 10.64898/2026.05.03.722518

Figure Lengend Snippet: (a) Schematic illustrating how BD shapes the in vivo immunostimulatory activity of self-dimerizing RNA-1 delivered by LungLNPs or LiverLNPs. LungLNP enhances delivery of RNA-1 to the lungs (1, pink), whereas conventional LiverLNP delivery directs RNA-1 to the liver (1, blue). In each case, organ-specific accumulation leads to uptake of RNA-1 into tissue resident immune or non-immune cell populations expressing pattern recognition receptors (PRRs) (2, pink/blue), thereby influencing pharmacodynamic responses, cytokine release, immune activation, and tumor suppression. (b) IFN-luciferase reporter assay in A549 IRF3 dual reporter cells showing induction by RNA-1 formulated in LungLNPs vs LiverLNPs, compared with free RNA-1 and empty controls. Data presented as average ± SD, n = 3. (c) Schematic of the in vivo pharmacodynamic (PD) model used to assess plasma cytokines following systemic administration of LungLNP/RNA-1, LiverLNP/RNA-1 formulations and corresponding empty LNPs. Mice were dosed with 2.2 mg/kg of RNA-1. (d-h) Quantification of peak plasma cytokine levels (2h for IFNα, IFNβ, TNFα and 6h for IFNγ, IFNλ), (i–m) Temporal kinetics of plasma cytokines (IFNα, IFNβ, IFNλ, IFNγ, and TNFα) following treatment at 2, 6 and 24 h. Data are represented as mean ± SEM from a representative experiment of three independent experiments with n = 6–7 (d–h) and n = 5–7 (i–m) biologically independent samples. (n) Schematic presentation depicting the knockout models used to study the innate immune pathway activated by LungLNPs/RNA-1 (2.2 mg/kg) in mice. (o) Quantification of IFNα plasma levels in RIG I KO mice (cytoplasmic sensing) compared with wildtype (WT) control. (p) Quantification of IFNα plasma levels in TLR3 and TLR7 KO mice compared with wildtype (WT) control. Data are represented as mean ± SD from a representative experiment of two independent experiments with n = 3-6 (o–p) biologically independent samples. (q) Molecular illustration depicting an Alphafold3 modeling of mouse RIG I and mouse TLR7 engaged with dsRNA-1 or ssRNA-1 respectively. Panels a, c and n were created with BioRender.com. The data were analyzed by ordinary one-way ANOVA with Tukey’s multiple-comparisons test; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: Human NF-κB-SEAP & IRF-Luc Reporter lung carcinoma (A549) cells (A549 RIG I) and human RIG-I-KO Dual Reporter A549 cells (A549 RIG I KO) (InvivoGen) were used to study the in vitro innate immune activity of RNA-1.

Techniques: In Vivo, Activity Assay, Expressing, Activation Assay, Luciferase, Reporter Assay, Clinical Proteomics, Knock-Out, Control

Journal: bioRxiv

Article Title: Enhanced lung delivery of an immunostimulatory duplex RNA augments the antitumor activity by reshaping systemic cytokine pharmacodynamics

doi: 10.64898/2026.05.03.722518

Figure Lengend Snippet: (a) Schematic illustration depicting a cross-section of the human lung cancer chip model, which recapitulates key physiological and pathophysiological features of human lung cancer. The microfluidic chip top channel containing human lung epithelial cells and human A549 adenocarcinoma alveolar basal epithelial cells stably expressing GFP, bottom channel containing human lung microvascular endothelial cells cultured on all four walls of the lower channel. (b) Treatment regimen for the human lung cancer-chip using LungLNPs/RNA-1 (100 and 200 nM), and empty LungLNP control (LungLNPs/Empty, 200 nM) and untreated chips. The first treatment was administered 4 days post-seeding, followed by establishment of the air–liquid interface on the same day. A second dose was administered on day 8. LNPs were delivered by vascular perfusion for 6 h per treatment. (c) A549 tumor growth curves during the treatment regimen, quantified by longitudinal GFP fluorescence imaging and measurement of fluorescence intensity. Data were analyzed using a two-way mixed effects model with time and treatment as fixed effects, followed by Tukey’s multiple-comparisons test. (d) Representative fluorescence images showing A549 tumor cells (green) on day 11 (scale bar = 1000 µm). (e) Quantification of cytokines and chemokines measured 2 h following the second dose. Data were analyzed by one way ANOVA with Tukey’s multiple comparisons test. (f) LNP uptake in the lung cancer-chip following perfusion of fluorescently labeled LungLNPs/RNA-1 Cy (yellow) at 100 and 200 nM. Endothelial cells were stained for VE-cadherin (purple), A549 tumor cells expressing GFP are shown in blue, and nuclei are shown in white. Chips were imaged 4 days post-treatment using confocal microscopy (scale bar = 20 µm). (g) Schematics depicting the mechanistic insight into RIG I-mediated lung cancer immunotherapy in human lung cancer chip demonstrating internalization into endothelial cells and RIG I activation and secretion of cytokines. Uptake into epithelial cells via direct exposure or via transport through gaps in the endothelial barrier. Panels a, b and d were created with BioRender.com. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: Human NF-κB-SEAP & IRF-Luc Reporter lung carcinoma (A549) cells (A549 RIG I) and human RIG-I-KO Dual Reporter A549 cells (A549 RIG I KO) (InvivoGen) were used to study the in vitro innate immune activity of RNA-1.

Techniques: Stable Transfection, Expressing, Cell Culture, Control, Fluorescence, Imaging, Labeling, Staining, Confocal Microscopy, Activation Assay