The ability to systemically administer siRNAs and functionally modulate gene expression in tissues of interest is considered by many an important step to opening up the therapeutic potential of RNAi to a wide range of diseases. The liver is arguably the best example where intravenously administered siRNAs have already been shown to potently knockdown target genes from mice to non-human primates.
The most promising delivery technologies so far (next to gene therapy vectors such as AAV which I will not discuss here) involve liposomes such as the SNALP particles which were first pioneered by Protiva scientists and subsequently became the subject of intense legal battles involving Protiva, Tekmira, Sirna Therapeutics, and Merck. While facilitating highly efficacious gene knockdown, toxicities such as liver enzyme elevations and non-linear relationships between knockdown and siRNA dosage have been repeatedly reported with these chemistries. While the toxicities were observed at relatively high dose levels, non-linear dose responses complicate the choice of the right dose for entering clinical trials.
One step in the right direction was taken when Daniel Anderson from the MIT reported earlier this year at the Keystone meeting the identification of a slightly different class of compounds which they termed “lipidoids”. Importantly, this class of chemistries appear to efficiently promote RNAi gene knockdown with little if any apparent toxicities and linear dose responses.
The just released paper by Rozema and colleagues from Mirus Bio Corporation (Rozema et al. PNAS Early Edition 24 July 2007: Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes) sheds light on some of the issues above and holds out a new paradigm for achieving safe and efficacious therapeutic RNAi knockdown in select cell populations of the liver. Rather than regarding drug delivery to the liver as a passive process given that the bulk of intravenously injected drugs will pass through it with a relatively high chance of entering resident cells there, the present approach involves attaching a simple galactose-derived ligand thereby actively targeting asialoglycoprotein receptors (ASGPr) displayed on hepatocytes.
Knockdown of ApoB100 and microscopic analysis of fluorescently tagged small double-stranded nucleic acids confirmed that hepatocytes were indeed efficiently transfected. Equally important, however, was their observation that Kupffer cells, a major population of macrophages in the liver that are implicated in many cases of drug-related liver toxicities, did not take up the siRNA mimicks, whereas non-ASGPr targeted particles were able to transfect surrounding cells, including Kupffer cells.
Taken together with the SNALP and lipidoid data, this study supports the hypothesis that Kupffer cells may act as a sink for certain siRNA formulations such as SNALPs, thereby not only causing non-linear dose responses, but also an immunogenic response and related toxicities. It also shows that it should be possible to design simple and small siRNA nanoparticles for RNAi delivery from relatively cheap materials. Although lipidoids appear to be a clinically viable technology already, it is comforting to know that alternative routes exist and gratifying to see almost daily improvements being made in the RNAi delivery field.
PS: An interesting aspect of this publication was data the authors obtained on ApoB knockdown. In addition to serving as a target for early proof-of-concept studies for systemic RNAi delivery, ApoB has been a favourite for treating hypercholesterolemia using both antisense and RNAi. ISIS Pharmaceuticals in particular has an anti-ApoB antisense compound in late phase II clinical trials. However, only a month ago, Alnylam reported that in their hands ApoB knockdown led to unacceptably high levels of fat accumulation in the liver (fatty liver phenotype), a finding that was confirmed in the present study. Moreover, both reports indicate that this was a siRNA sequence specific effect and was achieved using two different delivery methods. This physiologic response makes a lot of sense, since ApoB’s main role is in the export of cholesterol and triglycerides from the liver. Failure to export them should accumulate them in the liver.
This is a good example where RNAi can serve both as a target validation tool AND a platform for developing innovative drugs. Consequently, I fully support Alnylam’s decision to target PCSK9 in their hypercholesterolemia program instead, which is a genetically well validated target for reducing LDL-cholesterol and heart disease (see also Blogs from 6 May, 2007: “Preventing Heart Disease with RNAi Therapeutics”, and 9 May, 2007: “ISIS Copies Alnylam’s Heart Disease Strategy“). It is curious then that ISIS maintains and has published the absence of such a phenotype. For the sake of patients and stakeholders in ISIS, I can only hope they are right. The explanation for the apparent discrepancy? I cannot really offer a good one except to speculate on a fortuitous ISIS 301012 antisense off-target effect.
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