Despite the Nobel Prize-winning discovery of RNA interference (RNAi) over a decade ago and the billions of dollars spent developing therapeutic applications, delivery issues continue to challenge the field. To address these problems, researchers are creating novel strategies to achieve a safe and effective mode of delivery.
New approaches were highlighted at the “Oxford RNAi Conference” held last month in the U.K. and will also be presented at Select Biosciences’ “RNAi and miRNA World Congress” to be held later this month.
Emerging strategies include the use of nanoparticles and nanotubes as carriers for the short interfering RNAs (siRNAs) that deal the blow of gene knockdown via mRNA degradation. Scientists also are attempting to refine homing strategies for delivery utilizing the exquisite specificity of antibodies.
Xavier de Mollerat du Jeu, Ph.D., staff scientist at Life Technologies says there are three problems that need to be solved before siRNA advances to the clinic, “delivery, delivery, delivery”, referencing the well-known quote once made by gene-therapy pioneer Inder Verma, Ph.D. This has been and continues to be the key challenge.
The problem is that there are four barriers that a successful siRNA therapeutic must overcome. Once introduced into an organism, patrolling macrophages and cells of the reticuloendothelial system see the siRNA complex as foreign and attempt to degrade and eliminate it. A second important hurdle is the need to target the siRNA to the proper organ/cell of interest. Thirdly, once inside the cell the siRNA must be able to escape the endosome pathway that also attempts to degrade it. Finally, once the siRNA overcomes those obstacles, then it must be potent enough to accomplish knockdown.”
So far the most promising results in vivo were obtained with liposomes, according to Dr. de Mollerat du Jeu. “The field has achieved a great deal of success in utilizing siRNA in cultured cells. Translation to in vivo delivery has been more of a challenge. Our company has developed lipid-based nanoparticles for in vivo animal work. We recently launched Invivofectamine® 2.0 Reagent. After a single intravenous injection of 5 mg/kg FactorVII siRNAs complexed with Invivofectamine® 2.0, we observed 90 percent mRNA and protein level reduction in the liver for more than three weeks.”
There are other potential time and money-saving applications for this technology. “There are many uses, especially as a substitute for transgenic mice for target validation and ADME studies. Currently, researchers must develop expensive animal models that knockout a certain gene in order to determine its mechanism of action. However, the use of Invivofectamine with an appropriate siRNA could achieve the same results and also demonstrate the mechanism of action in much less time and at a fraction of the cost.”
Invivofectamine can multitask, says Dr. de Mollerat du Jeu. “Another useful feature of Invivofectamine is the ability to knock down up to four targets at once. This is a very powerful way to dissect whole pathways for assessing many interactions at once. For the future, we continue to create new applications for our reagent such as for liver cancer, and envision new uses by changing the route of delivery such as direct injection into the brain or tumors.”
Carbon Nanotube Delivery
Another siRNA nano-based delivery strategy is the use of long cylindrical fibers called carbon nanotubes. Interest in carbon nanotubes developed after the discovery that carbon could form stable and ordered structures other than diamonds and graphite. Thousands of papers have been published on these remarkable and versatile structures that range in diameter from the nanometer to micron range.
A key advantage of carbon nanotubes is that they can act like nanoneedles, easily piercing the cell membrane to deliver the goods, says Kostas Kostarelos, Ph.D., professor in the Nanomedicine Laboratory, Centre for Drug Delivery Research, The School of Pharmacy, University of London.
“There are fundamental differences between nanoparticles and carbon nanotubes. While spherical nanoparticles have been used in the last few decades, we are now finding that fiber-shaped nanotubes have dramatically different interactions with biological matter, such as the plasma membrane. In cell culture, carbon nanotubes can deliver siRNA directly into the cytoplasm with orders of magnitude higher levels than nanoparticles.”
“The challenge for biomedical applications has been the insolubility of carbon nanotubes in most buffers in general, and in biological fluids in particular. To overcome this they have been coated with amphiphilic molecules (lipids and polymers) or functionalized with various chemical groups that can vastly improve their water dispersibility. But the degree of aggregation and individualization of nanotube materials in biological fluids such as blood and interstitial fluids have important roles in their pharmacological performance.”
Other challenges also need addressing. “One caveat is that the gene knockdown we see is not commensurate with the amount of siRNA delivered inside the cell,” Dr. Kostarelos reports. “Our working hypothesis is that the siRNA is not being efficiently released from the nanotube. We and others are working to prepare constructs with improved detachment capabilities.”
Nanotube vectors are utilized by the Nanomedicine Lab for the local delivery of siRNAs in the central nervous system. “The beauty of this approach is that we can administer the therapy directly at the loci in the brain where it is needed. Think of it like the extension of a syringe at the nanoscale. In animal models we were able to rescue stroke-damaged brain tissue in mice using this approach. Other applications we are working on include Parkinson disease and brain cancers.
“We are still in the early stages of this technology, but hope our work will act as an impetus to further explore and, ultimately, clinically translate the therapeutic capacity of chemically functionalized carbon nanotubes.”