Over the years, researchers have demonstrated that RNAi compounds can be designed to specifically reduce the expression of target genes before their mRNA is translated into protein. By leveraging this knowledge, RNAi compounds can be developed into drugs to prevent overexpression of deleterious proteins in target cells, thus providing therapeutic benefit.
At GTC’s recent “RNAi Research and Therapeutics” conference, speakers presented their solutions to the challenges faced by clinical researchers looking to develop RNAi therapeutics. How do I deliver RNAi compounds to target cells? Do I need a delivery vehicle to facilitate cellular uptake? Can I demonstrate sufficient knockdown, either transiently or permanently, in my target cells?
RNAi screens provide an essential tool for gene discovery and validation of gene function. One of the preferred approaches for RNAi screening recommended by Thermo Fisher Scientific employs the use of pooled shRNAs. This approach is supported by protocols and a large portfolio of products, including shRNA libraries.
“The screening strategy of using pooled shRNAs provides a large collection of lentivirus-expressed shRNAs that target thousands of genes in a single well,” indicated Devin Leake, Ph.D., global director of R&D, molecular biology at Thermo.
“Cells with shRNAs targeting certain genes respond differently to phenotypic selection and become enriched or depleted during the screen. Identifying the exact shRNA producing the phenotype is then determined using next-gen sequencing (NGS) of genomic DNA isolated from the cells.”
Success depends on three factors: first, the use of high-performance shRNAs that effectively silence (knockdown) target gene expression; second, a high representation of shRNAs in the pool; and third, maintenance of the relative abundance of shRNA molecules during analysis.
Experimentally, Dr. Leake’s team has determined that providing the shRNAs in 500-fold excess yields the best, most consistent results. It is important to remember that in the context of the screen, there will be both high-abundant targets and low-abundant targets.
The shRNAs are packaged in lentiviral particles and delivered to the cells via the normal retroviral process. That means that the transduced shRNA can integrate into the cells’ genome, providing a permanent knockdown of the targeted gene.
Cells transduced with pools of shRNA can be treated with a particular therapeutic agent of interest and screened for specific phenotypes. Cells lacking a response indicate a wild-type phenotype suggesting that shRNAs in those cells target genes that are not involved in the disease state.
In contrast, cells that respond indicate that shRNAs target relevant genes to the disease state. In negative selection screens, shRNAs from responders will either be overrepresented in the final NGS analysis because of cell proliferation, or underrepresented because of cell death. Both outcomes help identify genes involved in the disease-state pathway and the role that they play during therapeutic intervention.
“The power of RNAi is enabling,” said Dr. Leake. “It is much faster and easier to make a knockdown than to create a gene knockout in either a cell line or in an animal model.”
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.