Once an siRNA and target gene of interest are identified, many researchers want to perform validation experiments in vivo . But many scientists would like to go directly in vivo using the same siRNAs that were used for in vitro experiments. In doing this, researchers have encountered some hurdles. The first was stability, but that problem has been largely solved. Several companies, including Alnylam Pharmaceuticals Inc., Cambridge, Mass., have developed modifications to siRNAs that make them more stable in plasma or serum. The first, the incorporation of a single phosphorothioate linkage at the 3' termini on each strand, protects the siRNA from exonuclease degradation; 2'-O-methyl and/or 2'-fluoro modifications can also be added to protect sites vulnerable to endonuclease cleavage, says Nagesh Mahanthappa, PhD, senior director of business development and strategy at Alnylam.
A more challenging obstacle to in vivo experimentation with siRNAs has been delivery. The penultimate goal would be to achieve a universal systemic delivery strategy for siRNAs. On this front, Mahanthappa says Alnylam has published that covalent conjugation of cholesterol to an siRNA molecule increases its circulating half-life and promotes uptake into a variety of cell types. Alnylam is also actively investigating other types of formulation approaches, such as mixing siRNAs with lipid components, to promote uptake by different cells.
That said, there doesn't seem to be a single, easy solution for systemic siRNA delivery, he says. "You're not going to see a one-size-fits-all strategy, even from a target validation perspective," says Mahanthappa. "I think there will be favored types of chemical modifications or formulations that will allow one to deliver siRNAs to particular cell types. As those are developed, it's going to give rise to a toolkit or a palate of methodologies which can then be applied on a case-by-case basis." >