Stable RNA Nanoscaffold May Solve SiRNA's Delivery Woes
BioWorld Today Science Editor
One of the biggest challenges of RNA interference remains its delivery. Getting siRNA into the cells where it's needed has been challenging on several fronts. For one thing, RNA is not the most stable molecule. For another, even when it is stable, how to get it into specific cells has been something of a head-scratcher.
But researchers at the University of Cincinnati think they may have found a way to solve both of those problems. By using a so-called three-way junction to bind different pieces of RNA together, they were able to make a highly stable scaffold that will stick together in the bloodstream. Each part of the scaffold can bind other molecules, such as targeting agents or therapeutic siRNAs.
The scaffold is based on so-called packaging RNA, a form of RNA that exists naturally in certain viruses. In those viruses, it forms a motor that the virus uses to package its DNA genome during replication.
Such motor proteins interlock tightly via so-called three-way junction motifs. And when they do, the resulting nanoparticle complex achieves a level of stability that is basically unheard of in the RNA world.
"The RNA nanoparticle . . . cannot be disrupted by regular reagents to disrupt normal DNA or RNA. It will not dissociate into piece after injected in the body with extremely low concentration," Peixuan Guo told BioWorld Today. "Dissociation of RNA nanoparticles in vivo is one of the barriers in RNA drug development," and the new nanoparticle makes the delivery of RNA or drug therapeutics in vivo "realistic." Guo is professor of biomedical engineering at the University of Cincinnati, and the senior author of the paper detailing the findings, which was published in the Sept. 11, 2011 , advance online issue of Nature Nanotechnology.
In their paper, Guo and his team first tried to separate the nanoparticles with detergents that are used in the lab to break down DNA and RNA. They found that the bonds between the subunits were strong enough to withstand detergent concentrations that will break normal DNA and RNA bonds.
The authors tested their three-way junction RNA in cancer cell lines, using a three-way junction RNA with each of the subunits fused to a different molecule. Many cancer cells overexpress receptors for folic acid, and so one subunit was fused to folate. Another carried the siRNA to silence surviving, an anti-apoptotic protein that allows cancer cells to escape cell death. The third was conjugated to a marker molecule.
When the authors delivered those RNAs to cell lines, they were able to bind the folate receptor and enter the tumor cells, where they reduced expression of survivin.
One of the challenges of RNA nanoparticles is that – like other compounds with only weak bonds – they tend to dissociate when they are at low concentrations in the bloodstream. To see whether the three-way junction nanoparticles would stay stable in the bloodstream, Guo and his team also tested them in animals with xenografted tumors. Here, too, folic acid- and marker-labeled RNAs were able to bind to tumor cells, but did not enter normal cells of the liver, heart and several other organs. They also did not enter muscle tissue.
Finally, the nanoparticles were much more stable in the bloodstream than regular siRNAs. Three-way junction RNAs stayed stable in the bloodstream from six to 12 hours after injection, while control siRNAs, the authors wrote in their paper, "could not be detected beyond 5 [minutes] post-injection."
Guo is a co-founder of West Lafayette, Ind.-based Kylin Therapeutics, which is using pRNA technology to develop cancer drugs. Kylin was founded in 2007 and had a Series A of $1 .2 million in 2008. The company's investors include In-Vivo Ventures, Golden Pine Ventures and Heartland Angels. The company also has grant support.
Published: September 26, 2011
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