Digging Deep To Understand siRNA Delivery Systems

When chemists get involved with siRNA delivery, they want to do more than just find new materials to take therapeutic nucleic acids to their targets. They want to understand how those materials work.

"The field of nucleic acid delivery has matured to the point that we need to be asking fundamental questions rather than empirical questions," says David Putnam, assistant professor of biomedical engineering at Cornell University.

Putnam, like many other chemists, is using materials originally developed for DNA delivery as a jumping-off point for finding new siRNA delivery agents. He is finding distinct differences in the behavior of DNA delivery polymers when used with siRNA compared with DNA. "The structures that work well for DNA delivery work only in a very narrow window for siRNA delivery," he says.

For example, polyethyleneimine (PEI) is used to deliver DNA over a wide range of formulations but is "much more finicky for siRNA delivery," Putnam says.

Putnam is now making libraries of polymers to determine their structure-activity relationships as delivery vehicles. He starts with a methacrylate backbone with side chains to which he can add a variety of other molecules such as sugars and lipids. "When you start doing combinations, asking how things work collectively, the number of materials you need to synthesize gets very large very fast," he says.

Kent Kirshenbaum, a chemistry professor at New York University, is also focusing on a material that has been shown to work with DNA. Kirshenbaum became interested in siRNA delivery agents when he wandered over to the neighboring lab of biology professor Fabio Piano. "It struck me immediately that we could develop improved chemical tools to solve their problems," Kirshenbaum says.

Kirshenbaum uses peptidomimetic oligomers called "lipitoids," which are conjugates of oligomeric cationic N-substituted glycines called "peptoids" and lipid head groups that can be independently modulated (Mol. BioSyst. 2006, 2, 312). Lipitoids were originally developed for DNA delivery by Ronald N. Zuckerman at Chiron Corp.

Kirshenbaum is interested in exploring the relationship between peptoid composition and the nature of the nanoparticles formed with siRNA. "We think there's a lot of really interesting questions to ask regarding the variation of the composition, the structure of the generated nanoparticles, and their ability to provide for cell uptake."

Kirshenbaum hopes to understand the physicochemical characteristics that give enhanced activity and then use that knowledge to generate a range of delivery reagents for siRNA.

"Our goal is to develop a platform that would allow us to create a library that could be used in different settings or for delivery to different cell types," he says. Such a library would allow him to mix and match off-the-shelf siRNAs and delivery agents.

Developing new siRNA delivery systems requires "a more sophisticated approach" than people have been using, Kirshenbaum says. "A big part of that is going to come from improving the understanding of siRNAs and chemical modifications to the RNA molecules, but I think there's also a huge opportunity for modulating the characteristics of delivery agents, as well."

A. James Mixson, a professor of molecular medicine at the University of Maryland, Baltimore, is focusing on so-called branched HK peptides, which are composed of histidines and lysines. Such peptides have been used in the past for DNA delivery. By increasing the ratio of histidine to lysine and increasing the number of branches from four to eight, Mixson's team can improve delivery of siRNA.

The lysine residues make the HK peptides positively charged at physiological pH, thereby allowing the peptides to complex the siRNA tightly enough to get into cells through endosomes. Once the siRNA-HK peptide complexes are inside the cells, the basic imidazole groups on the histidines buffer the endosomes, which are slightly acidic.

"The precise mechanism is not really known, but buffering plays an essential role in the lysis of the endosomes," Mixson says. "If we add a single additional lysine to each of the branches, it can dramatically decrease the siRNA delivery."

Mixson is currently working with Intradigm, an RNAi company in Rockville, Md., to develop the HK peptides as siRNA delivery agents by modifying them with polyethylene glycol and targeting ligands.

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