For small interfering RNA, approaching a cell is like walking up to the door of an old speakeasy. Such doors were heavily reinforced and had a narrow, built-in sliding panel at eye level, and if the eyes peering out though the open panel didn’t like the look of you, well, you were not getting inside. Failing to gain entry is something that happens all too frequently to small interfering RNAs, which admittedly are anything but “life of the party” types. These molecules are apt to suppress the expression of high-spirited proteins, helping subdue overly raucous proceedings—cancer, infectious processes, genetic disorders.
The calming effect is called RNA interference, or RNAi. It is a normal process that all cells use. It may also be instigated deliberately, therapeutically, through the introduction of small pieces of synthetic RNA, or specially designed small interfering RNAs (siRNAs).
Researchers have been using RNAi to inhibit the production of specific proteins that can cause disease if they are overly abundant or mutated. Each RNAi drug has a sequence that corresponds to the gene blueprint for a particular protein. Once inside the cell, the RNAi drug is loaded into an enzyme that specifically slices the messenger RNA encoding the target protein in half. This way, no protein is produced.
Unfortunately, due to their size and negatively charged phosphates on their backbone, RNAi drugs are repelled by the cellular membrane and cannot be delivered into cells without the help of a special delivery system. Besides, if siRNAs are left unadorned or unencapsulated, they are subject to degradation by nucleases in the bloodstream.
A delivery mechanism that is effective in cell culture involves packing siRNAs into viral vectors. But viral vectors can be risky in vivo because they are inherently immunogenic. An alternative approach is the use of lipid or synthetic nanoparticles as delivery vehicles. Large nanoparticles, however, tend to be taken up by the liver, which can complicate the targeting of other tissues. Also, nanoparticle-delivered siRNAs often undergo catabolism via the endosomal pathway.
Yet another approach is the use of molecular conjugates, chemically modified siRNAs. This is the approach championed by a group of researchers based at the University of California, San Diego. These researchers, led by Steven Dowdy, Ph.D., assert that they have developed modified siRNAs that may serve as RNAi prodrugs.
The researchers described their work November 17 in the journal Nature Biotechnology, in an article entitled, “Efficient delivery of RNAi prodrugs containing reversible charge-neutralizing phosphotriester backbone modifications.” In this article, the authors propose that siRNA delivery may be achieved by synthesis of short interfering ribonucleic neutrals (siRNNs) whose phosphate backbone contains neutral phosphotriester groups, allowing delivery across the plasma membrane.
“Once inside cells, siRNNs are converted by cytoplasmic thioesterases into native, charged phosphodiester-backbone siRNAs, which induce robust RNAi responses,” they wrote. “siRNNs have favorable drug-like properties, including high synthetic yields, serum stability and absence of innate immune responses.”
siRNNs, suggested Dr. Dowdy, are more subtle than nanoparticles: “From a molecular perspective nanoparticles are huge, some 5,000 times larger than the RNAi drug itself. Think of delivering a package into your house by having an 18-wheeler truck drive it through your living room wall—that’s nanoparticles carrying standard RNAi drugs. Now think of a package being slipped through the mail slot—that’s siRNNs.”
In the Nature Biotechnology article, Dr. Dowdy and colleagues indicated that siRNNs had more specific advantages: “Unlike siRNAs, siRNNs avidly bind serum albumin to positively influence pharmacokinetic properties. Systemic delivery of siRNNs conjugated to a hepatocyte-specific targeting domain induced extended dose-dependent in vivo RNAi responses in mice.”
The siRNN technology described in the article is being developed by Solstice Biologics, a biotech company co-founded by Dr. Dowdy. Another co-founder, Curt Bradshaw, Ph.D., serves as the company’s chief scientific officer.
Solstice hopes to refine the mechanism of prodrug release by introducing new structural features. Already, Solstice reports that it has enhanced performance characteristics such as deliverability, potency, stability, and ease of synthesis.
“Our most recent advances are now being directed to deliver nucleic acid therapeutic to non-liver tissues,” indicated Dr. Bradshaw.
Although delivery challenges have frustrated attempts to develop RNAi drugs, researchers remain hopeful that they can help realize RNAi’s potential to selectively disrupt biological pathways that cause disease. Another motivation is RNAi’s unique adaptability. As cancer and viral genes mutate, RNAi drugs can be easily evolved to target them. This allows RNAi therapy to keep pace with the genetics of the disease—something that no other type of therapy can do.