Almost daily, there is important research being published documenting the progress towards making RNAi Therapeutics become a reality. In addition, I am again and again surprised by the revelations of key regulatory roles of the natural counterparts of the RNAi triggers, the microRNAs, in many important diseases which lays the foundation for a healthy microRNA Therapeutics industry in its own right. In my next two postings, I would therefore like to highlight two such quite nice stories published recently, one about broadening the potential of SNALP RNAi delivery technology by understanding its molecular biological basis (this posting), the other about microRNA-33 as an exciting new target for increasing the ‘good’ HDL-cholesterol (next posting).
Towards Actively Targeted SNALP Delivery
The liver, however, is not where it stops. Solid cancers have emerged as another major opportunity for SNALP for which today’s technology may already be adequate for a number of indications. Due to the flexibility and inherent potential of the platform, however, there is much room to increase the important therapeutic window not only for solid cancers, but also for other indications such as inflammation, infection, and hematologic abnormalities. The Holy Grail towards this is ligand-targeted delivery which also requires increasing SNALP circulation times partly by limiting their uptake in the liver and making them invisible to undesired phagocytic cells ('stealth').
A paper by by Akinc and colleagues at Alnylam points towards how this might be achieved. In an important first step, the apolipoprotein E was (ApoE) was found to be critical for the functional delivery of basic, i.e. not actively targeted ionizable SNALP liposomes (iSNALP). Note here that SNALPs typically come in 2 basic shapes: those that are positively charged at physiologic pH (‘cationic SNALPs’ to which e.g. the ‘lipidoid’-containing SNALPs belong to), and those that are neutrally charged at physiologic pH, but that become positively charged at acidic pH (‘ionizable SNALPs’ which Tekmira focuses on, while Alnylam develops both types). SNALPs of the latter kind have a number of characteristics that make them preferable for most systemic RNAi applications. One is that the positive charge makes them inherently more 'sticky' biologically which is not only detrimental to their pharmacology, but can also cause a number of toxicities, including the damage of cell membranes. iSNALPs, by contrast, do not suffer from these drawbacks.
On the other hand, positive charge is important for efficient encapsulation of the siRNAs into the liposomes and also beneficial for cell transfection by first interacting with the negatively charged cell exterior and subsequently disrupting the endosomal lipid bilayer. Because iSNALPs become positively charged at low pH (hence 'ionizable'), they can still be efficiently formulated by transiently decreasing the pH during packaging, and they can still efficiently escape from the acidifying endosome into the cytoplasm for the same reason. One outstanding question, however, has been how iSNALPs, for which the potencies now approach the single digit microgram/kg range (about 100-fold more potent than 1st generation SNALPs and about 1000-fold more potent than RNaseH antisense), make critical contact with target cells in the first place without the positive charge.
Employing an impressive array of genetic cell and animal models, Akinc and colleagues show that it is the plasma protein Apolipoprotein E (apoE) that allows, by associating with the lipid layer of the SNALP, the iSNALP particles to recognize cells carrying cognate receptors of the LDL-receptor family. Confirming the distinct uptake pathways involved in cationic and iSNALP delivery, the ApoE-LDLR interaction was not rate-limiting for cationic SNALP delivery. As a side note, because the LDL-R itself seems to be the major receptor in the liver, this needs to be taken into account when using iSNALPs for the homozygous familial hypercholesterolemia population in the upcoming SNALP hypercholesterolemia clinical trials.
Hints that ApoE could be involved in iSNALP delivery actually came from earlier studies on neutral liposomes furthermore illustrating how the wealth from decade-long prior liposomal research is now benefitting the development of the SNALP delivery platform.
In order to demonstrate ApoE-independent targeted delivery of iSNALPs, the researchers then decided to increase the stability and density of the exterior PEG layer, generally used as a stealth strategy, such that ApoE would not be able to easily associate with the lipid layer of the particles, and also to increase the circulation times such that iSNALPs would have sufficient time to distribute throughout the body. To replace ApoE’s function in cellular uptake, another ligand could now be added, in this case to the end of the PEG-lipids, such that they re-enabled uptake, but in an ApoE/LDL-R independent manner.
Despite this fundamental demonstration of re-targeting iSNALPs, the main challenge now is to do so without losing knockdown potency. In the present study, increasing the stability and density of the PEG layer decreased knockdown potency by about a log, from the sub 0.1ug/kg now routinely achieved with ApoE-targeted iSNALPs to close to 1mg/kg, probably due to either of the following 2 factors: 1) despite cell surface receptor recognition of the particles, the dense pegylation inhibited subsequent endosomal uptake; 2) the re-targeted iSNALPs were taken up, but then remained inert inside the endosomes because of inefficient shedding of the PEG layer thus leaving the endosomolytic lipids unexposed.
By using techniques such as fluorescence microscopy, it should be fairly straight-forward to figure out which one of the two possibilities it is, if not a combination. Whatever it is, optimizing the stability and density of iSNALP pegylation, the location of the targeting ligands (outside on the PEG and/or on the lipid bilayer), and exploiting alternative mechanisms of shedding the PEG layer (e.g. through the incorporation of proteolytic cleavage sites or pH-dependent shedding), should be just some of the approaches that should solve these challenges, and may indeed differ depending on the disease indication. Moreover, in practice it may not be necessary to completely abrogate ApoE-mediated uptake to sufficiently re-target iSNALPs.
I am sometimes reminded, also by readers of this blog, that there are also other siRNA delivery technologies besides SNALP. That’s certainly true, and some of them have shown encouraging data indeed, including in humans, and are important components of the RNAi technology landscape as RNAi Therapeutics will ultimately involve the use of more than one delivery platform. On the other hand, there is little arguing that the development of SNALP technology in the last 6 years has been breathtaking and of the highest scientific quality and its success should be to the benefit of the entire RNAi Therapeutics space.
Milestones in SNALP siRNA delivery
2005: SNALP delivery in rodents; recognition of innate immune stimulation
2006: SNALP delivery in non-human primates with EC50s of around 1mg/kg
2007: siRNA modification abrogates SNALP-siRNA triggered TLR immune stimulation
2009: SNALP delivery for (non-liver) metastatic solid cancer; demonstration of RNAi mechanism of action
2010: SNALP potency approaching single-digit microgram/kg potencies
2010: biological basis for SNALP delivery being elucidated, laying the foundation for increasingly wider applications of the technology
Looking forward to your post on miR-33. Today we find out that microRNA 140 acts against arthritis progression. This is apparently the first evidence that non-coding RNA plays a key role in age-dependent diseases.
ReplyDeleteDo you see Tekmira's SNALP being used to create microRNA mimics and inhibitors in the near future?
ReplyDeleteHow would SNALP compare to other delivery systems for creating safe and effective microRNA mimics and inhibitors? Too early to predict?
ReplyDeleteSNALP certainly has great potential for the delivery of microRNA mimics. Think cancer. It remains to be seen what the utility of SNALP would be for the delivery of microRNA inhibitors. Difficult to say whether the pharmacologies are suitable and it would also depend on the microRNA inhibitor structure and mechanism of action, but I can imagine that in circumstances where you desire fast delivery of the inhibitors, e.g. in the case of acute viral infections, SNALP delivery of microRNA inhibitors could also have its uses.
ReplyDeleteIn terms of how SNALP might compare to other types of microRNA mimic and inhibitor delivery methods, at least for the mimics, the comparison would not be too different from the relative deliveries of RNAi triggers. For microRNA inhibitors again, the comparison is probably not as simple and would depend on the specific therapeutic goals. Sorry for being so vague.