5'ppp-dsrna control lyovec  (InvivoGen)

Journal: bioRxiv

Article Title: Template-Directed RIG-I Agonist Assembly for Targeted Cancer Immunotherapy

doi: 10.1101/2022.12.08.519592

Figure Lengend Snippet: a) Design of the mir-21-template directed RIG-I agonist, ss-ppp-miRNA-21. The antisense ss-miRNA-21 oligonucleotide, designated as ss-ppp-miRNA-21, was designed to be fully complementary to the target miRNA-21 and to incorporate a 5’ triphosphate modification; b) Biogenesis and function of miRNA (black solid arrow). Starting in the nucleus, imperfectly or perfectly complementary miRNA:miRNA* duplexes are produced in the cytoplasm, where they are incorporated into the Argonaute-containing miRNA induced silencing complex (miRISC), unwound, and the mature miRNA strand retained in miRISC, while the complementary strand is released and degraded. miRISC is guided by the mature miRNA, which directs it to complementary sites in target mRNAs, resulting in translational repression and/or mRNA degradation; c) Activation of RIG-I signaling pathway (red dashed arrow). Upon entry into the cell, ss-ppp-miRNA-21 competes effectively with endogenous mRNA targets (mostly only partially complementary) to bind to the miRNA in the miRISC complex, resulting in release from the RISC and formation of a 5’-ppp blunt ended double stranded RNA. The blunt-ended 5’ppp-dsRNA can be readily captured by RIG-I and trigger its activation. RIG-I signaling in the tumor cells leads to type I IFN-driven immune response and preferential activation of programmed tumor cell death, releasing various immunological factors into the TME; d) Activation of cell-mediated immunity (dark red solid/dashed arrow). RIG-I signaling promotes rapid rise of type I IFNs and direct cancer cell death, releasing IFNs and pro-inflammatory cytokines as well as tumor antigens (TAs). Both innate immune cells and the adaptive immune system are energized in the remodeled TME. Leukocytes such as NK cells and macrophages boost their cytolytic activity in response to an IFN-rich environment. Importantly, an increase in IFNs and TAs triggers an adaptive immune response, resulting in the maturation and activation of macrophages and DCs, and enhanced antigen presentation to T-lymphocytes in tumor draining lymph nodes. Naïve T cells are then activated, propagated, differentiated, and transported to the TME, where they can perform an effective anti-tumor response by direct or indirect cytolytic activity mediated by perforin and granzymes, and the secretion of cytokines like IFNγ and TNFα. Following cancer cell death, the majority of tumor-specific effector CD8+ T cells die via apoptosis, but some survive to mature into long-lived protective memory CD8+ T cells. RIG-I, retinoic acid induced gene 1; IFN, interferon; NK, natural killer; DC, dendritic cell; TME, tumor microenvironment; TA, tumor antigen; APC, antigen-presenting cell; PRR, pattern-recognition receptor; TNFα, tumor necrosis factor alpha; Treg, T-regulatory cells. (Parts of the figure were drawn by using pictures from Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license).

Article Snippet: Cells (3x10 5 ) were transfected with 1 μg/mL, ds-ppp-RNA positive control (InvivoGen), ss-ppp-miRNA-21 (8 μg/mL), or ss-miRNA-21 (4 μg/mL) with or without miRNA-21 mimic (1000 ng/mL), using LyoVec transfection agent (Catalog Code: lyec-12, InvivoGen).

Techniques: Modification, Produced, Activation Assay, Activity Assay

Journal: bioRxiv

Article Title: Template-Directed RIG-I Agonist Assembly for Targeted Cancer Immunotherapy

doi: 10.1101/2022.12.08.519592

Figure Lengend Snippet: a) RIG-I protein expression in HEK-Lucia™ RIG-I cells. The whole cell lysates were collected and assayed (50 μg) for the RIG-I proteins by Western analysis using specific antibody. HEK-Lucia™ RIG-I cells were found to express high levels of RIG-I protein. b) RIG-I activation by ds-ppp-RNA (+ive control) in HEK-Lucia™ RIG-I and HEK-Lucia™ Null control cells. Cells were transfected with Lyovec control or stated concentrations of ds-ppp-RNA with Lyovec. Luciferase activity was measured in supernatants after 48 h as per manufacturer’s recommendations. The commercially available ds-ppp-RNA was shown to robustly induce RIG-I activation (n = 3; *, p < 0.05; **, p < 0.01). c) Comparison of ss-ppp-miRNA-21 and ss-miRNA-21-mediated dose-dependent RIG-I activation in Null vs RIG-I expressing reporter cell lines. HEK293 RIG-I cells or Null cells were treated with the indicated concentrations of agonists. Luciferase activity was measured in supernatants after 48 h. At all tested concentrations, luciferase activity was significantly higher in the HEK-Lucia™ RIG-I cells than in the HEK-Lucia™ Null cells, indicating RIG-I specific activation. Both the ss-ppp-miRNA-21 and ss-miRNA-21 designs led to RIG-I activation (n = 3; *, p < 0.05; **, p < 0.01). d) Relative expression of miRNA-21 in B16-F10 cells. B16-F10 cells express moderate levels of miR-21, as compared to a reference cell line (murine breast 4t1), known to express abundant miR-21 (n = 3; *, p < 0.05; **, p < 0.01). e) Caspase-3/7 activation (dose dependent) by ppp-ss-miRNA-21 in B16-F10 cells. Cells were transfected with varying concentrations of ppp-ss-miRNA-21 or ss-miRNA-21 with Lyovec. After 48 h cell death was measured by a CaspaseGlo assay. ss-ppp-miRNA-21 induced a dose-dependent increase in caspase activation that was not observed when using ss-miRNA-21 (n = 3, p ≤ 0.05). f) Cell viability reduction (dose dependent) by ss-ppp-miR21 in B16-F10 cells. Cells were transfected with varying concentrations of ss-ppp-miRNA-21 or ss-miRNA-21. Cell viability was determined using CellTiter-Glo® 2.0 Assay after 48 h of incubation. There was a dose-dependent decrease in tumor cell viability in the presence of ss-ppp-miRNA-21 but not ss-miRNA (n = 3; p ≤ 0.05).

Article Snippet: Cells (3x10 5 ) were transfected with 1 μg/mL, ds-ppp-RNA positive control (InvivoGen), ss-ppp-miRNA-21 (8 μg/mL), or ss-miRNA-21 (4 μg/mL) with or without miRNA-21 mimic (1000 ng/mL), using LyoVec transfection agent (Catalog Code: lyec-12, InvivoGen).

Techniques: Expressing, Western Blot, Activation Assay, Transfection, Luciferase, Activity Assay, Comparison, Caspase-Glo Assay, Incubation