Lipidoids Expand Chemical Space for Cationic Liposome Delivery of RNAi Therapeutics

[Important update, see end of entry]

Following a long series of presentations and publications involving Protiva, Tekmira, Alnylam, and Merck/Sirna, PEG-stabilized cationic liposomes known as SNALPs have to be considered one if not THE most advanced systemic RNAi delivery technology to date. While efficacy was very potent at single-digit mg/kg doses, the major drawback of the first studies on SNALP-siRNA delivery were slight elevations in liver enzymes, an indicator of toxicity (Zimmermann et al. study). It was therefore important to expand on those studies and search for formulations with even better knockdown efficacies and inherently less toxicity, thus pushing the therapeutic index well into predictably clinically safe ranges.

The recent announcements 0.1mg/kg IC50 knockdown efficiencies for SNALP-like formulations by both Protiva and Tekmira (due to their upcoming merger from now on referred to as Tekmira for simplicity) support the notion that the exploration of new chemistries should facilitate the development of SNALP RNAi for clinical use, and although only the surface has been scratched, a first IND with realistic chances at therapeutic success may not be very far away.

To exploit the chemical space available for SNALPs though requires the ability to generate new and diverse lipid chemistries as well as the ability to manufacture and formulate these chemistries to scale. The latter has been achieved by Tekmira’s spontaneous vesicle formation by ethanol dilution method allowing for the speedy manufacture of SNALP-siRNA formulations that can support late pre-clinical and clinical studies, while the long-awaited paper by Akinc and colleagues from the MIT (Langer/Anderson lab) and Alnylam Pharmaceuticals on so called lipidoids and that has now been published in Nature Biotechnology [Akinc et al. (2008): A combinatorial library of lipid-like materials for delivery of RNAi therapeutics.], will now faciliate the efficient exploration of novel, SNALP-compatible lipid chemistries.

Rather than laboriously synthesizing and testing one lipid after the other on a hypothesis-driven basis, Akinc and colleagues developed a synthesis method that allowed them to generate libraries of cationic lipids with quite unusual and diverse characteristics to systematically evaluate them for siRNA delivery. A first library gave an indication of which chemistries worked better than others, and a second library was generated based on the characteristics of the best performing ones in the first set.

Initial tests were based on silencing reporter genes in tissue culture. It should be noted that for these high-throughput experiments, probably for speed and ease, simple siRNA-lipoplexes were used (siRNA-lipid mix), instead liposomally formulated siRNA as for later tests in vivo (siRNA captured inside liposomes), and this may be one limitation as it could have caused them to miss even more promising in vivo silencing chemistries. In any case, the best candidates were then taken forward into rodent and non-human primate studies, this time formulating the lipids together with cholesterol and PEG-lipid into cationic liposomes, essentially based on the same principles as SNALPs (stabilized liposomes containing diffusible PEG-lipids, the latter interestingly manufactured by Alnylam itself).

Overall, IC50s in the low mg/kg range were routinely observed for a number of liver targets. This was achieved without significant toxicities based on careful safety analysis, and only in some cases mild elevations, less than 2-fold, of liver enzymes were observed. Similarly, the absence of negative interference with endogenous microRNA pathways was reported last year. This is a good start and may not have employed the most efficacious siRNAs, but by optimizing the formulations further, e.g. by engineering additional fusogenic lipids and other properties into these particles, sub-mg/kg doses that would be desirable in the clinic should be achievable and still be compatible with the more scalable manufacturing technologies practiced by Tekmira (although 50nm particles and high encapsulation efficiencies were achieved, the extrusion-based method as employed in the present study may limit scale).

Beyond the liver, the lipidoid formulation showed some promise for the delivery of siRNAs to the lung as demonstrated in an RSV model. Interestingly, inhibition of RSV replication was enhanced by lipidoid-siRNAs (almost 3-log knockdown at 2mg/kg) over unformulated, naked siRNAs (1-log knockdown), in contrast to previous studies on which Alnylam’s ALN-RSV01 is based that showed somewhat less viral knockdown with naked siRNAs and that suggested no enhancement of siRNA delivery to the lung by formulation with other delivery chemistries. In addition, given the propensity of such nanoparticles to be taken up by phagocytic cells of the immune system and the largely unmodified siRNAs used for targeting RSV, follow-up studies need to look at any innate immune responses elicited by such lipidoid-siRNA combinations.

In summary, this study opens up a wide chemical space for the systematic evaluation of cationic liposome-mediated delivery of drugs, particularly siRNAs, but also microRNA antagonists (demonstrated in this study) and beyond. Following some of the recent breakthroughs and due to the triangular relationship between Alnylam, Tekmira, and the MIT, complementary in terms of both know-how and IP, progress of SNALP-siRNAs into the clinic may hopefully occur within the next few months and should be followed by next-generation chemistries.

[Update May 1, 2008: According to a report by RNAiNews , an Alnylam spokesperson indicated that the company had not given guidance on the specific liposome formulations to be used for their hypercholesterolemia and liver cancer clinical programs. This is in contrast to an earlier report by RNAiNews from last year’s Beyond Genome conference in San Francisco which indicated that Alnylam had chosen “choose lipidoids over SNALPs” for these indications (also discussed in a blog entry here). It therefore seems that Protiva/Tekmira's 0.1mg/kg IC50 liposomal nanoparticle formulations may be the current frontrunners in entering the clinic (see also a recent PR).

The sometimes imprecise use of the terms SNALPs and lipidoids may be partly to blame for the confusion. SNALP refers to a liposomal formulation technique, while lipidoids are a new class of lipids generated by combinatorial chemistry which can now be evaluated for liposomal drug delivery. As such, lipidoids could be used within the context of SNALPs, and both of these approaches are therefore complementary to each other.]