Xavier de Mollerat du Jeu, Ph.D., senior scientist in the R&D transfection group at Life Technologies, is directing the effort to improve the current set of delivery vehicles used in life science research to deliver siRNA molecules in vivo. The ultimate goal is to develop a delivery vehicle that will enable therapeutic drug delivery.
While the company cut its teeth on transfection reagents that enabled nucleic acid delivery into primary cells and established cell cultures, a completely new formulation is required for efficient siRNA delivery in vivo. The optimal reagent, called Invivofectamine® 2.0, is derived from different lipids and has been shown to deliver siRNA molecules into hepatocytes in the liver following tail vein injections in mice.
Since the launch, the team has discovered new lipids and has been working on optimizing the ratios of the different components as well as mixing parameters. These improvements have resulted in a greater than 50-fold improvement in efficacy; ED50 levels have been reduced to 0.02 mg/kg. This has been a big step in moving from a great tool for research to an effective tool for therapeutics.
These liponanoparticles (LNPs) are not toxic based on liver tox and interferon testing. They are resistant to blood nucleases and significantly smaller than in vitro transfection reagents, so that they can pass through capillaries and into the cellular matrix.
“As a tool provider, we provide the clinical researcher with our Invivofectamine 2.0 reagent as an empty delivery vehicle. We also provide researchers with a protocol to encapsulate their siRNA molecule of interest. This delivery reagent works best for encapsulation of siRNA, miRNA, miRNA mimics, and miRNA antimirs,” indicated Dr. de Mollerat du Jeu.
“With intravenous injection in the mouse tail vein, these loaded particles deliver to the liver and facilitate uptake in hepatocytes. We recommend the use of Factor VII as a positive control for this system. Factor VII is synthesized in the liver and secreted into the bloodstream. Efficacy of delivery and gene expression knockdown is easily monitored by the measurement of Factor VII protein levels from a drop of blood taken from treated vs. untreated mice.
“To reach other organ systems, alternative routes of injection may be required. Preliminary results indicate that intraperitoneal injections enable delivery to the pancreas and the spleen, intracranial injections enable access to the brain, and intraorbital injections enable access to the eye. We have not yet tried it, but we believe that inhalation delivery should enable access to targets in the lung.”
The team is also working on alternative formulations that might facilitate delivery to organs outside the liver following IV injection. For this effort, different lipid formulations are being rationally developed based on all the design elements, including particle size, overall charge, and the ratio of each of the different components in the LNP. Analytical methods are used in vitro to screen for the best candidates. A subset of candidates are then tested in vivo using fluorescent-labeled siRNAs as probes to monitor sites of accumulation and cellular uptake.