Fig. 1: Construction and characterization of the genetic circuits. | Cell Research

Fig. 1: Construction and characterization of the genetic circuits.

From: In vivo self-assembled small RNAs as a new generation of RNAi therapeutics

Fig. 1

a Schematic description of the architecture of the genetic circuit. The core circuit contains a promoter part (black) and an siRNA-expressing part (red). When the core circuit is placed in a cell/tissue chassis, the promoter drives the transcription of the siRNA and directs the siRNA payload into exosomes. Two composable, plug-in parts are driven by the same promoter of the core circuit and incorporated into the core circuit structure: one encodes a guidance tag (green) that is anchored on the exosome surface to enable targeting of exosomes to cells and tissues, and the other part (blue) expresses a different tandem siRNA. The core circuit design facilitates the systematic distribution of siRNAs to multiple tissues, while the composable-core circuit increases the simultaneous accumulation of two siRNAs in specific tissues. b Quantitative RT-PCR analysis of the levels of the desired guide strand and undesired passenger strand generated using the pre-miRNA strategy (EGFR siRNA is embedded in pre-miR-155 and is controlled by a CMV promoter, named CMV-siRE) or the shRNA strategy (EGFR siRNA is placed in an shRNA and is controlled by a U6 promoter, named U6-shRE) in HEK293T cells. A CMV- or U6-directed scrambled RNA (CMV-scrR or U6-scrR) was used as a control. RNU6A (U6) was selected as a reference gene for normalization of cellular siRNA levels (n = 3 in each group). c Quantitative RT-PCR analysis of EGFR siRNA levels in exosomes derived from HEK293T cells transfected with the CMV-scrR or CMV-siRE circuit. miR-16, a universally expressed miRNA, was selected as a reference gene for the normalization of siRNA levels in exosomes (n = 3 in each group). d Quantitative RT-PCR analysis of EGFR mRNA levels in LLC cells treated with exosomes derived from HEK293T cells transfected with CMV-scrR or CMV-siRE (n = 3 in each group). Different doses (total protein contents of exosomes were 4, 20 and 100 μg, respectively) of exosomes were added to reveal the dose-dependent effect. e Western blot analysis of EGFR protein levels in LLC cells treated with exosomes derived from HEK293T cells transfected with CMV-scrR or CMV-siRE. Different doses of exosomes were added to reveal the dose-dependent effect. Left panel, representative western blots. Right panel, quantitative analysis (n = 3 in each group). f A CMV-directed Flag-Lamp2b fusion construct (CMV-Flag-Lamp2b) or a CMV-directed Lamp2b construct (CMV-Lamp2b) was transfected into HEK293T cells. Exosomes were then isolated and either directly loaded for western blotting with anti-Flag and anti-CD63 antibodies (equal CD63 band densities indicate similar exosome levels) or immunoprecipitated with IgG or anti-Flag beads before western blotting. g The CMV-siRE, CMV-siRT or CMV-siRE+T circuits were transfected into HEK293T cells. A quantitative RT-PCR assay was performed to assess the levels of EGFR and TNC siRNAs in transfected HEK293T cells (n = 3 in each group). h CMV-scrR, CMV-siRE or CMV-Flag-siRE+T circuits were transfected into HEK293T cells. Exosomes were then isolated and either directly loaded for western blotting with anti-Flag and anti-CD63 antibodies or immunoprecipitated with IgG or anti-Flag beads before western blotting. A quantitative RT-PCR assay was performed to assess the levels of EGFR and TNC siRNAs in immunoprecipitated exosomes (n = 3 in each group). i Quantitative RT-PCR analysis of EGFR and TNC mRNA levels in U87MG cells treated with exosomes derived from HEK293T cells transfected with the CMV-scrR, CMV-siRE or CMV-RVG-siRE+T circuit (n = 3 in each group). Different doses of exosomes were added to reveal the dose-dependent effect. j Western blot analysis of EGFR and TNC protein levels in U87MG cells treated with exosomes derived from HEK293T cells transfected with the CMV-scrR, CMV-siRE or CMV-RVG-siRE+T circuit. Different doses of exosomes were added to reveal the dose-dependent effect. Left panel, representative western blots. Right panel, quantitative analysis (n = 3 in each group). Values are presented as the means ± SEM. Significance was determined using one-way ANOVA followed by Dunnett’s multiple comparison in d, e, g, i, j. *P < 0.05; **P < 0.01; ***P < 0.005; ns, not significant.

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