RNAi research has capitalized on the ability to design and deliver small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) to knock down expression of target genes with strong selectivity and specificity.
These genetic tools have applications in many research areas to study the genetic mechanisms and functions of various pathologies, leading to the potential for therapeutic applications.
Life Technologies has developed tools for the design and research applications of siRNAs, explains senior staff scientist Alexander Vlassov, Ph.D. The Silencer® Select platform includes thousands of siRNAs with LNA chemical modifications and optimal designs to target all human, mouse, and rat genes in highly potent and specific fashion. Customers can also submit their own sequences to design custom siRNAs for unusual targets, or if they want to test additional siRNA designs for a particular target. Silencer Select siRNAs are a good option for in vitro experiments, says Dr. Vlassov.
The Ambion® In Vivo siRNAs, developed specifically for in vivo applications based on the parental Silencer Select platform, have proven to be highly stable when injected into the bloodstream of animal models, he continues. Although in contact with nucleases found in body fluids, these siRNAs do not readily degrade due to stabilizing chemical modifications.
The half-life of Ambion In Vivo siRNAs in the bloodstream is more than 24 hours, while regular siRNAs can degrade in a matter of minutes, Dr. Vlassov notes. By using these stable siRNAs, researchers can achieve prolonged effects and perform fewer animal injections. He explains that with a single injection at a low dose, the duration of siRNA-mediated knockdown can last for up to one month.
Along with siRNAs, Life Technologies has also developed tools for miRNA research. Similar to siRNAs, miRNAs downregulate gene expression, but usually do so by repressing translation instead of inducing mRNA cleavage.
miRNA mimics are 18–25 bp double-stranded synthetic molecules (with limited chemical modifications) that mimic endogenous miRNAs and are used to increase their intracellular levels, while miRNA inhibitors are single-stranded oligonucleotides (with heavy chemical modifications) that bind to a miRNA of interest and reduce its intracellular levels.
When using an miRNA mimic or inhibitor, readouts of knockdown or derepression, respectively, of mRNA or protein levels of the miRNA’s targets can confirm the efficiency of the mimic or inhibitor. Life Technologies’ offers mirVana™ products to target miR-122 in vivo, and miR-1 and let-7 in vitro, along with a number of reporter assays.
Dr. Vlassov says that the library of miRNA mimics and inhibitors is frequently updated to match the latest version of miRBase, the online miRNA database. Additionally, he notes that “due to the natural complexity of miRNA pathways, there may be a risk of off-target effects because miRNAs do not have perfect homology to their targets, each miRNA can have hundreds of targets, and every mRNA can be targeted by multiple miRNAs. It’s a complicated story, but our products are still highly specific despite these challenges.”
Life Technologies provides a panel of lipid-based delivery reagents, such as Lipofectamine® RNAiMAX and Invivofectamine® 2.0, for transfection in cell culture or animal models, respectively. While efficient delivery of siRNA or miRNA cargo into cells is usually straightforward, in vivo systemic delivery can be quite challenging and presents more barriers to overcome, such as the potential for some material to become bound to blood proteins or be filtered out by the kidneys before reaching the organ of interest. Currently, the Invivofectamine 2.0 reagent can effectively deliver siRNA and miRNA cargo into liver cells in animal models, says Dr. Vlassov.
“We would like to develop separate Invivofectamine reagents to transfect siRNAs into the lungs, brain, spleen, and kidney, as it remains difficult to consistently and effectively transport siRNA cargo to these tissues via tail vein injection.”
Issues of siRNA specificity have been identified in a number of studies over the last few years, in which siRNA delivery results in phenotypes that can only be explained by deregulating genes that were not intended for targeting by the siRNA, explains Michael Hannus, Ph.D., who works at the University of Regensburg and collaborates with Intana Bioscience. Dr. Hannus indicates that these off-target effects can be quite extensive, as one siRNA design can potentially target 8,000 human genes on average.
Reducing Off-Target Effects
“Our approach was to design multiple siRNAs that all have the same ‘on’ target but all have different ‘off’ targets,” Dr. Hannus remarks. “With a sufficiently complex siRNA pool, the on-target gene knockdown effect will remain strong while the off-target effects of each individual siRNA are diluted with an increasing number of siRNA designs contained by the pool.”
By spiking in a control siRNA previously published as demonstrating strong off-target effects, Dr. Hannus and his collaborators confirmed that the number of deregulated genes was dramatically reduced by increasing the complexity of the siRNA pool, as measured on an Affymetrix genomic microarray. Furthermore, the researchers discovered that a pool of approximately 30 siRNAs provided sufficient complexity necessary to substantially dilute any off-target effects.
According to Dr. Hannus, “Our technology provides complex but defined siRNA pools. Using an algorithm, we select a collection of optimal sequences to compose the pool.” The research team developed a novel enzymatic approach to generate siRNA pools of defined sequences via in vitro transcription and a specific nuclease. All siRNAs are precisely 21 nucleotides long to provide maximum efficiency and avoid any possible effects from the interferon response.
Dr. Hannus and colleagues are currently working on expanding their collection of siRNA pools, termed “siPools.” The goal is to assemble a genome-wide human siPool library within the next two years. Furthermore, as the enzymatic approach is far cheaper than chemically synthesizing siRNAs, large amounts of siPools can be produced at a low cost, offering an attractive alternative for in vivo RNAi applications.
Polymer-Based Delivery of siRNAs
As an alternative to cationic lipid-based delivery, Dynamic Polyconjugates™ (DPCs) are polymer-based vehicles for delivery of siRNAs into specific tissues or cell types of interest to promote efficient and stable knockdown, explains Dave Rozema, Ph.D., vp of chemistry at Arrowhead Research.
DPCs are 5–20 nm in size and are composed of a polymer to which targeting ligands are reversibly attached. The DPCs are guided to the cell of interest by the targeting ligand and are taken up by the endosome, which is lysed by the polymer to release the siRNA cargo into the cytoplasm. Dr. Rozema noted that using polymer-based technology allows for easy control of the size of the resulting formulation, whereas nucleic acid–lipid complexes tend to have a larger size that may make delivery more difficult.
When DPCs were first developed by Arrowhead, the siRNA cargo was covalently attached to the polymer (hence the term “polyconjugate”), precluding the need for electrostatics to hold the two together, as is the case for lipid-based delivery.
“We have recently discovered that it is not necessary to attach the two if the siRNA and polymer each have their own targeting groups; they will still meet up in the targeted tissue by co-injection without being physically attached to one another,” Dr. Rozema explains. “There may be a marginal difference in efficacy with respect to the siRNA dose, but what we gain is a huge simplicity in the formulation because we can make the siRNA and polymer separately, so for manufacturing and analytics it becomes a much easier task.”
Arrowhead is pursuing this style of DPC formulation with intravenous delivery of siRNA-mediated knockdown of hepatitis B virus, in which the modified polymers and siRNA cargo are targeted to hepatocytes. Arrowhead is also researching DPC designs for subcutaneous liver delivery, as well as identifying high-capacity, rapidly internalizing ligands for targeting to other tissues.