Invivofectamine® 2.0 Reagent is the latest innovation for in vivo siRNA delivery from Life Technologies. Invivofectamine® 2.0 Reagent efficiently delivers siRNA in vivo through systemic delivery, producing highly effective mRNA, protein and functional knockdown. The reagent demonstrates a low toxicity profile and is exceptionally easy to use.
Invivofectamine® 2.0 Reagent is a breakthrough for in vivo RNAi delivery.
- Easier to use than any other reagent for in vivo siRNA delivery- mix, equilibrate, and inject
- Provides specific and effective knockdown (80% or greater knockdown) in liver
- Phenotypic knockdown is directly correlated to target knockdown
- The complex of Invivofectamine 2.0 Reagent and siRNA is stable
- Intravenous injection targets liver tissue
- The complex demonstrates extremely low toxicity
Creating complexes of Invivofectamine® 2.0 Reagent and RNAi duplexes for delivery is easy (Figure 1). Simply mix, equilibrate, and inject.
Complexes comprising Invivofectamine® 2.0 Reagent and Ambion® in vivo siRNA targeting Factor VII have been successfully delivered by tail vein injection to liver tissue (Figure 2). We have demonstrated effective knockdown of Factor VII at the mRNA level (Figure 2A) and by loss of protein activity in serum 2 days post injection (Figure 2B).
RNAi-mediated knockdown occurs through siRNA interaction with the RISC complex resulting in a site-specific cleavage of the target mRNA. The cleavage site can be detected by using a method known as RACE (Rapid Amplification of cDNA Ends). The data shown in Figure 2C verifies that the knockdown observed was mediated through the RISC complex and is specific to the siRNA target sequence.
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Figure 2. Use of Invivofectamine® 2.0 Reagent Results in Targeted Knockdown in Liver After a Single Intravenous Injection.
Invivofectamine® 2.0 Reagent complexed with siRNA targeting Factor VII mRNA (injected at doses of 1, 3, 5, and 7 mg/kg achieved >95% knockdown of target mRNA levels (A) (knockdown was assessed by TaqMan® assays), and >95% reduction in protein activity (B) (blood serum was isolated and assayed for Factor VII activity 48 hours post injection via a chromogenic substrate).The knockdown observed was RNAi-mediated (C) as shown by detection of the siRNA-induced mRNA cleavage fragment by RNA ligase-mediated rapid amplification of 5' cDNA ends (5′ RLM-RACE). Schematic depicting the location of the predicted Factor VII-specific siRNA cleavage site and the primers used for PCR amplification of the cleavage fragment are shown above. 5′ RLM-RACE PCR amplification products from treated mice were of the expected size (208 bp band) on agarose gels.
Gene expression knockdown in vivo results in expected phenotypic changes
Apolipoprotein B (APOB) is the primary apolipoprotein of low-density lipoprotein (LDL), which is responsible for carrying cholesterol to tissues. Accordingly, studies of reduced expression of APOB have shown decreased levels of cholesterol and triglyceride. With high efficiency of knockdown, Invivofectamine® 2.0 Reagent complexed with siRNA targeting APOB has demonstrated specific reduction in levels of both cholesterol and triglyceride (Figure 3). Specificity and effectiveness of Invivofectamine® 2.0 Reagent gives researchers confidence that the observed phenotypes are attributed to specific target knockdown and not to non-specific toxicity.
Figure 3. Invivofectamine® Reagent and siRNA targeting APOB demonstrate knockdown in liver after a single intravenous injection.
Invivofectamine® 2.0 Reagent complexed with APOB siRNA was injected at dose of 3, 5, and 7 mg/kg. (A) Invivofectamine® 2.0 Reagent complexed with APOB siRNA achieves a greater than 95% knockdown in mRNA and approximately an 80% knockdown in protein levels. Blood serum was isolated and assayed for cholesterol (B) and triglyceride content (C) and the results show that the knockdown of ApoB protein resulted in a significant reduction in cholesterol, LDL, and triglycerides.
Knockdown duration is dependant on dose and stability of the siRNA molecule
A single injection of Invivofectamine® 2.0-siRNA complex results in a dose-dependant duration of knockdown at the protein level that can extend to 36 days. Duration of knockdown can also be affected by the stability of the siRNA molecule. We injected Invivofectamine® 2.0 Reagent complexed with the same sequence of siRNA, but one with the Ambion® In Vivo siRNA modifications and the other without modification at the same dose. Figure 4 shows that the increased stability of the Ambion® In Vivo siRNA modifications resulted in an extended duration of knockdown as compared to the unmodified molecule.
Figure 4. Knockdown Duration is Dependant on Dose and Stability of the siRNA Molecule.
(A) A dose-specific study of various concentrations of siRNA targeting Factor VII was performed. siRNA molecules were complexed with Invivofectamine® 2.0 Reagent and injected at doses of 3, 5, and 7 mg/kg. Serum was collected on days 2, 9, 15, 22, and 29 post injection and analyzed for protein knockdown (FVII) by a chromogenic assay. (B) Modified and unmodified siRNA molecules were complexed with Invivofectamine® 2.0 Reagent and injected at a dose of 7mg/kg. Serum was collected at various time points and analyzed for protein knockdown (FVII) by a chromogenic assay. CTRL: control; FVII: Factor VII
The biodistribution of Alexa Fluor® 647-labeled siRNAi is displayed in Figure 5. As expected, most of the siRNA was delivered to liver cells after intravenous injection; however, we did detect some delivery to the spleen and kidney. In the kidney, cryosectioning revealed that the labeled siRNA was delivered to the glomeruli. Interestingly, a different biodistribution was observed after intraperitoneal injection. We observed delivery of labeled siRNA to spleen and pancreas cells, as well as in the liver, although to a lesser degree than intravenous injection.
Figure 5. Biodistribution of Alexa Fluor® 647-labeled Stealth RNAi™ siRNA Complexed with Invivofectamine® 2.0 Reagent.
Tissues were harvested 4 hours after intravenous or intraperitoneal injection of Alexa Fluor® 647-labeled Stealth RNAi™ siRNA complexed with Invivofectamine® 2.0 Reagent (7 mg/kg), and single cells from each tissue were analyzed by flow cytometry.
To evaluate in vivo toxicity of Invivofectamine® 2.0 Reagent, several detailed studies focusing on analysis of a blood chemistry panel, hematology, and cytokine levels at multiple time points post injection were performed.
Figure 6. Invivofectamine® 2.0 Reagent is Nontoxic up to 10 mg/kg. Toxicity analysis was performed using Invivofectamine® 2.0 Reagent complexed with negative control siRNA. siRNA complexed with Invivofectamine® 2.0 Reagent was injected at 5 and 10 mg/kg, 24 and 48 hours after intravenous injection, serum was collected and toxicity markers analyzed. ALB: Albumin; ALP: Alkaline phosphatase; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; BUN: Blood Urea Nitrogen; CA: Calcium; CL: Chloride; GLOB: globulin; GLU: Glucose; K: potassium; NA: Sodium; PHOS: phosphate; TBIL: total bilirubin; TPRO: total protein
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