Tuesday, September 18, 2007

Journal Club: Alnylam and Collaborators Make Progress in Understanding and Optimising siRNA Uptake In Vivo

In yet another elegant paper, Alnylam and collaborators from the ETH in Zurich and the Rockefeller report this week in the journal Nature Biotech on the mechanism of siRNA uptake in vivo (Wolfrum et al.). This is significant, since a systematic approach to understanding siRNA delivery in vivo should most adequately address the delivery challenge that is considered by many to be the main barrier to the broad application of RNAi as a therapy. It is also a sign of the maturity of the RNAi field in general that no only are there now a variety of innovative delivery systems evaluated almost by the day, systems that show promise serve as leads for a detailed investigation of the underlying biology.

The study by Wolfrum and colleagues follows another high-profile publication 3 years ago (Soutschek et al.) where Alnylam scientists demonstrated gene silencing in mice following systemic administration of cholesterol-conjugated siRNAs. That study showed that although such siRNAs could silence genes particularly in the liver and gut, quite high amounts of siRNAs were needed (50mg/kg). By studying the uptake of the siRNA conjugates in these tissues, the authors not only hoped to understand why they functioned at all, but also to optimise their potency.

Efficient in vivo drug delivery requires favourable pharmacokinetics. Particularly, a drug has to be present in the blood for sufficient length of time so that it has a chance to accumulate in its target tissue. One reason for example why many experimental drugs fail is because they are rapidly excreted through the kidneys. This may often be prevented if the drug could interact with components of the blood such as the abundant lipoprotein particles.

Indeed, the authors find that siRNAs conjugated to cholesterol or other lipophilic molecules associated with the similarly greasy HDL and LDL lipoprotein particles. These would ferry them around in circulation and bring them into the proximity of cells that carry on their surface receptors for either HDL and/or LDL. Strikingly, pre-assembling the siRNA with purified HDL and LDL particles quite significantly increased the potency of the siRNAs. Furthermore, mice lacking either of the receptors for the lipoproteins were much less prone to gene silencing by the same pre-formulated siRNA particles.

In a further interesting twist, it was shown that siRNAs were not taken up by the cells as part of internalising lipoproteins, but that the siRNAs would take advantage of their proximity to the cell membrane during the docking, release, and re-docking process of their lipoprotein carriers with their receptors. Amazingly, through a combination of gene knockdown experimentation and blockage by antibodies, at least one of the actual entry routes for the siRNA was inferred to be the human homologue of the SID-1 gene that had earlier been shown to mediate systemic RNAi in the worm C. elegans.

Systemic RNAi describes the spread of an siRNA from one cell to another cell in the same or even different tissues. Systemic RNAi in worms and plants is associated with the amplification of RNAi, and both systemic RNAi as well as RNAi amplification were thought to have been lost during human evolution. It is therefore a surprise that SID-1 would still function in siRNA uptake, with demonstrated selectivity for siRNAs relative to other types of nucleic acids. This also raises the intriguing possibility that some sort of natural siRNA uptake should occur in humans.

Of more immediate importance, the present paper opens the door for the systematic screening of new lipophile-siRNA conjugates with improved association kinetics with lipoprotein particles, or even pre-formulation of such conjugates with lipoproteins or other natural or synthetic carriers of the blood. I look forward to what this line of investigation will yield next.

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By Dirk Haussecker. All rights reserved.

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