How do you get short RNAs into mammalian cells? The question has puzzled scientists since Andrew Fire and Craig Mello demonstrated the concept of RNAi in 1998. Short interfering (si)RNAs can jam the signals from specific genes, thus providing promise for exquisite genetic-based therapies. The challenges include aiming for the right mechanism without dragging other pathways into the fray, bypassing immune reactions, and simply getting past the cell membrane.
In dealing with living organisms, researchers had to start at the membrane. "Our main concern was the difficulty of getting highly charged nucleic acid inside the cell to reach the protein with the targeted activity," says Martin Woodle of Intradigm based in Rockville, Md.
Breaking the Barrier
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Staying on Target
Just getting into the cells is only the first challenge. Ratna Ray, a professor in the pathology department of St. Louis University, says she worries about the toxicity of siRNA at high levels. Brian Silverman, of Lerner Research Institute in Ohio, says, "You might get interferon response in vivo," which would lead to upregulation of interferon-stimulated genes. Delivering siRNAs can potentially activate nonspecific inflammatory responses, but Lieberman says her group's repeated experiments show that the fusion protein-delivery system targets only intended cells. "If any drug can target a specific cell, that reduces the toxicity of drug, and the overall dose would be lower," says Lieberman. Ray says she hopes the nanoparticle will provide a slow release system of siRNA, reducing chances of toxicity.
Both teams still need to determine whether these siRNA therapies will have off-target effects. Aimee Jackson of Rosetta Inpharmatics published an important warning in 2003.
Woodle says the problem with studies showing off-target effects is that all experiments to this point have been in vitro and therefore inadequate to predict siRNA behavior in animal models, and especially in people. Scaria and Woodle say the mouse model showed minimal siRNA interaction in other VEGF pathways and no physiologic side effects. Woodle is confident that precise targeting of cells will make the approach viable. "The objective of the nanoparticle system is to limit biodistribution to target tissue," says Woodle. "It's the right place to be exploring a solution."