Breakthrough in vivo transfection reagent
Invivofectamine® 3.0 Reagent is a breakthrough reagent for in vivo RNAi delivery, with greatly improved performance and up to 85% knockdown achieved using microgram levels of siRNA. View the Invivofectamine® 3.0 FAQs
Liver Bid suppression for treatment of fibrosis associated withnon-alcoholic steatohepatitis
Akiko Eguchi1, Xavier De Mollerat Du Jeu2, Casey D. Johnson1, Andronikou Nektaria2, Ariel E. Feldstein1
1Department of Pediatrics, University of California – San Diego, 9500 Gilman Drive, La Jolla, USA; 2Life Technologies Corporation, Carlsbad, USA
Easy to use
Creating complexes of Invivofectamine® 3.0 Reagent and RNAi duplexes for delivery is easy: simply mix, incubate (30 min), dilute, and inject (Figure 1).
Figure 1. An injection-ready RNAi/Invivofectamine® 3.0 Reagent complex can be prepared quickly and in just a few steps.
Effective, targeted knockdown
Complexes of Invivofectamine® 3.0 Reagent and Ambion® in vivo siRNA targeting Factor VII or Stealth™ RNAi PPIB have been successfully delivered by mouse tail vein injection to liver tissue (Figure 2). We have demonstrated effective knockdown of Factor VII and PPIB at the mRNA level.
Figure 2. Use of Invivofectamine® 3.0 Reagent enables targeted knockdown in the liver after a single intravenous injection. Invivofectamine® 3.0 Reagent complexed with siRNA targeting mRNA for Factor VII (FVII) or PPIB, injected at doses of 1 mg per kilogram mouse body weight (mg/kg), achieved as much as 85% knockdown of target mRNA levels (knockdown assessed via TaqMan® assay).
Less siRNA required to achieve effective knockdown
Complexes of Invivofectamine® 3.0 Reagent and siRNA in a range of amounts were introduced via tail vein injection. FVII protein levels in the serum were measured using a chromogenic assay 24 hours after injection (Figure 3). The amount of knockdown is correlated with the amount of siRNA in the complex. The ED50 of Invivofectamine® 3.0 Reagent is 0.1 mg/kg, compared to previous levels of 1.0 mg/kg.
Figure 3. Invivofectamine® 3.0 Reagent and siRNA targeting FVII produce dose-response knockdown in liver after a single intravenous injection. Invivofectamine® 3.0 Reagent complexed with Ambion® in vivo siRNA targeting FVII was injected at doses ranging from 0.02 to 2 mg/kg. Blood serum was isolated and assayed for FVII protein levels (Biophen® chromogenic assay).
Sustained knockdown after a single injection
A single injection of an Invivofectamine® 3.0 Reagent/siRNA complex results in significant knockdown at day 1 and for up to 3 weeks (Figure 4). Higher amounts of siRNA in the injected complexes resulted in longer-lasting knockdown over the range tested.
Figure 4. Study of dosage effect using three concentrations of siRNA targeting Factor VII (FVII). siRNA molecules were complexed with Invivofectamine® 3.0 Reagent and injected at doses of 1, 0.5, and 0.25 mg/kg. Serum was collected on days 2, 5, 9, 16, 23, and 30, and serum was analyzed for FVII protein knockdown by a chromogenic assay.
Gene expression knockdown in vivo results in expected phenotypic changes
Apolipoprotein B (ApoB) is the primary apolipoprotein of low-density lipoprotein (LDL), which carries cholesterol to tissues. Accordingly, studies monitoring reduced expression of ApoB have shown decreased levels of cholesterol and triglyceride. With high-efficiency knockdown, Invivofectamine® 3.0 Reagent complexed with siRNA targeting ApoB has demonstrated specific reduction in levels of both cholesterol and triglyceride (Figure 5). The specificity and effectiveness of Invivofectamine® 3.0 Reagent helps give researchers confidence that the observed phenotypes are attributed to specific target knockdown and not to nonspecific toxicity.
Figure 5. Invivofectamine® 3.0 Reagent and siRNA targeting ApoB achieve knockdown in liver after a single intravenous injection. Invivofectamine® 3.0 Reagent complexed with ApoB siRNA was injected at doses of 1.5, 0.5, and 0.25 mg/kg. Delivery of the complex containing the dosage of 1.5 mg/kg siRNA resulted in >80% knockdown in mRNA and ~70% knockdown in protein levels. Blood serum was isolated and assayed for cholesterol and LDL content, and the results show that the knockdown of ApoB protein resulted in a significant reduction in cholesterol and LDL.
Stability of Invivofectamine® 3.0 Reagent after thawing
The recommended long-term storage temperature for Invivofectamine® 3.0 Reagent is –20°C, but we have demonstrated that Invivofectamine® 3.0 Reagent can be stored at 4°C for at least 14 days after thawing (Figure 6), allowing you to extend your experiment over several days without needing to refreeze the reagent. If desired, however, the reagent can be frozen and thawed up to 4 times without loss of performance (data not shown).
Figure 6. Invivofectamine® 3.0 Reagent stored at –20°C can be thawed and kept at 4°C for up to 14 days. Invivofectamine® 3.0 Reagent was thawed and complexed with FVII siRNA and injected intravenously at 0.125 and 0.5 mg/kg doses. Aliquots of the same thawed reagent were used to create complexes for subsequent injections at 6 and 14 days after the first injection. After each siRNA injection, blood was collected and FVII protein levels evaluated (Biophen® chromogenic assay). These results demonstrate that thawed Invivofectamine® 3.0 Reagent stored at 4°C can be used for up to 14 days with no reduction in performance.
Low in vivo toxicity
To evaluate in vivo toxicity of Invivofectamine® 3.0 Reagent, blood chemistry analysis and cytokine measurements were performed at multiple time points following injection of the reagent (Figures 7 and 8). The results show that the levels of the assayed biomarkers in individuals transfected using Invivofectamine® 3.0 Reagent were not significantly different from those that did not receive the reagent.
Figure 7. Blood chemistry analysis of samples from mice injected with Invivofectamine® 3.0 Reagent. Invivofectamine® 3.0 Reagent complexed with Ambion® in vivo siRNA targeting FVII was injected into mice at doses of 1 mg/kg , 3 mg/kg , or untreated [U]. Blood samples were collected at 2, 24, and 48 hr and were evaluated using clinical chemistry assays for several biomarkers (Antech): glucose (GLU, in mg/dL), alkaline phosphatase (ALP, in U/L), alanine aminotransferase (ALT, in U/L), aspartate aminotransferase (AST, in U/L), total bilirubin (TBIL, in mg/dL), cholesterol (CHOL, in mg/dL), and triglycerides (TRIG, in mg/dL). Each bar comprises the data from four replicates.
Figure 8. Cytokine analysis of samples from mice injected with Invivofectamine® 3.0 Reagent. Invivofectamine® 3.0 Reagent complexed with Ambion® in vivo siRNA targeting FVII was injected at a dosage of 0.25 mg/kg (with and without the addition of dexamethasone (Dex)). Following injection, blood samples were collected at 2, 6, 24, and 48 hr, and cytokine levels were measured using a multiplexed bead-based immunoassay kit. As a control, samples from individuals not treated with the reagent were subjected to the same cytokine panel assay. Each bar comprises the data from three replicates.