Let’s Go to San Francisco: Trends from the 4th RNAi Research and Therapeutics Conference

Still looking for an interesting RNAi Therapeutics conference to attend? The 4^th RNAi Research&Therapeutics conference to be held in San Francisco June 20-21 may be a good choice to satisfy your cravings for not only RNAi Therapeutics research and development, but also the wider applications of RNAi gene silencing for target identification and new interesting trends in targeting non-coding RNAs.

Register with discount code ‘RNABLG13’ for 20% discount and free RNAi Therapeutics blog T-shirt here.

Reading through the abstracts of conferences like this one is helpful in understanding the trends in a scientific field.  As an appetizer, the following will highlight a few of those promising scientific directions, challenges, and future investment flows as seen through the lens of an RNAi Therapeutics nerd.

Delivery, delivery, delivery

An old headline, but as the key value-driving effort, delivery naturally is a focus of an RNAi Therapeutics conference.  Importantly, the two most advanced and promising systemic delivery technologies, SNALP/LNPs and DPCs, are represented.  Pieter Cullis, an academician with ties to the Tekmira, AlCana, and Alnylam love triangle, will be talking about Tekmira-type LNPs.  Interestingly, while these LNPs are known to have applications for knocking down genes in the liver, solid tumors, and potentially sites of inflammation and lung epithelia in aerosolized form, the abstract notes their use in the central nervous system (CNS) for serious neurological disorders.  Of course, the CNS is the organ with many of the major unaddressed medical needs, so I am curious whether there will be actual data showing good distribution and efficacy of LNPs in this organ.  While I consider the CNS challenging in terms of achieving wide distribution with direct administered nanoparticle-based delivery technologies, the immune challenges of nanoparticles, including complement activation and phagocytic uptake, might be lessened in this organ.

David Lewis from Arrowhead Research will present the latest about the exciting DPC delivery technology, including data from the chronic HBV program (ARC520).  By then, IND-enabling tox studies for ARC520 should be completed and the first DPC-related IND filed.  His talk should also be of interest to those not as obsessed about DPCs as I am as he will speak about some of the mechanistic insights of the key endosomal escape process, insights which should be more broadly applicable.

Trinna Cuellar from Genentech will be speaking about ‘A systematic evaluation of antibody-mediated siRNA delivery and silencing’.  It seems that Genentech hopes to create RNAi Therapeutics value based on its expertise in monoclonal antibody pharmacology.  The talk should elucidate whether the strategy envisions only minimal antibody-siRNA conjugates, or whether it further embraces other delivery platforms.  Of note, Tekmira once mentioned that it had a technology evaluation with Genentech on antibody-targeted SNALPs and made the bold prediction that ‘Roche will be back’.

Curiously, not one but three presentations concern aptamer-targeted RNAi trigger delivery (note: aptamers are antibody-like nucleic acids).  We have had a number of reports of such aptamer-siRNAs, especially for cancer and HIV infection.  However, these reports have encountered much disbelief, especially since they lacked an explanation for their postulated endosomal escape.  With credible knockdown activity reported with the GalNAc-siRNA conjugates by Alnylam, aptamer-RNAi triggers should be similarly gaining in credibility, especially when combined with self-delivering RNAi trigger chemistries.  An experiment that I would like to see as part of the field regaining credibility is an ASGPR-targeted aptamer-RNAi trigger conjugate (ASGPR is the receptor targeted by the GalNAc sugar).  In addition to building confidence in the approach, it would also be an alternative to GalNAc-RNAi triggers should the sugar moiety cause injection site reactions in humans.

Some of the aptamer-RNAi trigger presentations also touch on the subject of bifunctional RNAi Therapeutics, meaning RNAi Therapeutics formulations with a second non-RNAi therapeutic activity.  These secondary activities may include receptor blocking or toll-like receptor (TLR) activation as in a talk on CpG-STAT3 siRNAs by Hua Yu from the City of Hope.

In ddRNAi it is about the RNAi trigger design

The conference also includes presentations on microRNA processing and their implications for ddRNAi trigger design.  Unlike in RNAi Therapeutics induced by synthetic RNAi triggers, RNAi trigger design remains a crucial value-creating activity in DNA-directed RNAi Therapeutics.  Unfortunately, its importance has been entirely lost from the commercial field (if you really want to aggravate me, show me a U6-driven shRNA being put into clinical development).  Fortunately, academic efforts mean that ddRNAi trigger design continues to advance.

In his Keynote address, Mark Kay from Stanford University will present insights from his lab on microRNA processing and, crucially, how that knowledge can be put into practice for ddRNAi Therapeutics.  Especially recent work from Shuo Gu in his lab on Argonaute-specificity, strand selection, and Dicer processing have advanced this field and should also provide for fresh ddRNAi IP opportunities.  A presentation by Marco Weinberg from the Scripps Research Institute meanwhile is focused on an earlier step in microRNA processing, the primary microRNA recognition and cleavage by the Drosha enzyme.  ddRNAi trigger cassettes requiring the Drosha processing step promise to be more natural and cell-specific alternatives.  

MicroRNAs hot, lncRNAs getting hot

Functionally closely related to RNAi, it is not surprising that the conference program includes microRNA Therapeutics.  As by far the most advanced microRNA Therapeutics program, the update on the anti-miR122 program by Santaris for HCV infection (phase II) will be a highlight for those that have not been paying close attention to the results.  For those more familiar with the story, it will be of interest how Santaris aims to position this innovative treatment approach in an increasingly crowded and competitive field.

There will also be presentations on long non-coding RNAs (lncRNAs) and their therapeutic use.  LncRNAs are RNAs that do not code for proteins and are usually expressed in between protein-coding genes or antisense to or just upstream of protein-coding genes.  As more and more examples of such functional non-coding RNAs emerge, a whole cottage industry has emerged to cater to them.  Interestingly, reports suggest that lncRNAs can often be suppressed by RNAi triggers.  This is a departure from the classical thought that RNAi triggers can only target protein-coding mRNAs.  Similar to microRNA antagonists, targeting lncRNAs that silence other genes, can activate gene expression.  Oligonucleotide research reagent companies have naturally recognized the commercial potential of this by offering large lncRNA-targeting RNAi trigger libraries and Zaklina Strezoska will present the design approach by Thermo Fisher.

RNAi trigger libraries are also the focus of presentations by Michael McManus from UCSF and Iain Fraser from the NIH.  These researchers employ the libraries for understanding biological processes, an effort that is also suitable for new drug target identification.  The library approach by McManus is unique in that every gene is targeted by many (~25) shRNAs.  This is done to increase the confidence that hits concern real targets and not just some off-target noise.  The approach is critically facilitated by next-generation sequencing (NGS).

Next-generation sequencing is also the focus of a late afternoon session at the conference.  Since NGS is ubiquitous in today’s research, keeping up-to-date on technological advances in this field is part of the homework of any RNAi Therapeutics buff.

The conference will finish on a Friday.  If you like to digest all the information and be inspired by nature at the same time, I recommend that you stay the weekend and rent a car to see some redwoods, coastline, and villages inhabited by eccentrics north of the Golden Gate Bridge.  Good weather guaranteed.

Register with discount code ‘RNABLG13’ for 20% discount and free RNAi Therapeutics blog T-shirt here

RNAi Therapeutics Trends from the International Liposome Society Meeting 2009

Christmas to me means going to London to learn about what liposomes have in store for RNAi Therapeutics. As you know, liposomes have emerged as the leading platform for the systemic delivery of RNAi Therapeutics and offers considerable promise for diseases of the liver, solid cancers, and after that potentially enhanced vaccines, infectious disease and immune cell-related disorders. The fact that liposomal drugs are on the market already should further speed up its development, not least from a regulatory point of view.

The liposome field appears to be split between between cancer and vaccines about 2:1. Especially the success story of doxil (liposomal doxorubicin) has shaped the field and should partly explain why it is attracted so much towards solid cancers for RNAi Therapeutics. The EPR effect, targeted delivery, tumor vascular biology have been recurring topics and research using various advanced imaging modalities nicely informs the application of liposomal siRNAs in oncology. For example, liposomes (not necessarily unique in that regard) are not uniformly distributed within the tumor, but form a concentration gradient around the blood vessels. Strategies to achieve more uniform drug delivery may involve metronomic dosing in which the drug is administered more gradually at lower dosages such that the simultaneous onset of cell killing in proximity to the vessels will relieve intratumoral pressure so that subsequently arriving liposomes are freer to move within the tissue. Hyperthermia or low-dose TNF-alpha were two suggested strategies for increasing the leakiness of tumor vasculature, also to improve liposomal tumor distribution.

Since the highest concentration is achieved around the tumor vasculature and because there are differences between normal and tumor vasculature, targeting genes within the endothelial and supporting cells has become an attractive anti-tumor strategy. This for example is also the strategy that Silence Therapeutics is pursuing and the lipoplex formulation (siRNAs associated with liposome on the outside, not inside as in a SNALP) for some reason might have its application here.

But once within the tumor, mostly the result of the overwhelming EPR effect, the question becomes how to get the liposome into cells and then release the drug cargo into the cytoplasm in the case of siRNAs. Ligand-targeted liposomes are one answer to increase the cellular uptake of the liposomes. Antibodies are an increasingly popular means (immunoliposomes), although they present certain manufacturing challenges. It is still debated how PEG affects cellular attachment. Some claim that you cannot take up a pegylated liposome, some say you can. The answer may be that it depends on the size of the PEG. This is important for example in the context of a targeted liposome and whether to add the ligand to the PEG or directly incorporate it into the lipid bilayer as a lipid-conjugate, and when and how the PEG should leave the liposome. For efficacy and safety, triggerability of membrane lysis is of course important. Too much charge for example can be cytolytic, so you would like to expose membrane lytic activities in a pH-dependent (endosomal acidification), redox or protease-dependent manner. For the liposome field, this typically means pH-sensitive lipids.

Combined chemo-siRNA therapeutics is another trend that will become important for liposomal cancer RNAi Therapeutics. Combining ever more chemotherapeutics appears to be a game of diminishing returns with triple chemo Rx often not any better than double chemo Rx. This is because of limited mechanism of actions of chemotherapeutics and associated resistance mechanisms. RNAi Therapeutics therefore can either target these resistance pathways directly or bring to the table an entirely new anti-tumor mechanism of action.

I have always found it attractive to apply siRNAs for developing more effective vaccines. While I am not familiar with the literature around virosomes, viral ghost shells, a presentation by Swiss company Pevion Biotech caught my attention in this context. Basically, they are using flu viruses, disassemble them to get rid of the viral RNA and associate protein, and then reassemble them. During the reassembly process, they can then variably add their lipid-anchored antigen of choice and use the re-formulated virus as a vaccine. The fact that our bodies have seen flu virus before apparently helps to obtain a more favorable immunological response. By this, they believe to have broken the potency-toxicity linear correlation that seems to plague adjuvant science. While promising and based on a credible scientific presentation, it still does not appear to be among the most potent adjuvants, so I wonder whether an siRNA targeting the right immunomodulatory gene loaded into the virosome, essentially replacing the viral RNA there, could improve this equation even further. A challenge for nanoparticle-formulated siRNAs for enhanced vaccines is that although they are easily taken up by phagocyotic antigen-presenting cells, the phagocytic pathway usually destroys the siRNA and little escapes into the cytoplasm. The virosome could offer a solution since the flu virus is THE paradigm of how membranes fuse with each other and also explains how an extracellular antigen, the vaccine, can produce a favorable cellular immune reponses in this system. What I noted at the conference is the surprising lack of cross-talk between people using liposomes for vaccines versus those using them for oligonucleotide delivery which is unfortunate in cases like virosomes.

Pevion also seems to be producing good-quality liposomes. For liposomal RNAi Therapeutics, it is probably making the jump from rodents to primates and scalable manufacturing that separates the boys from the men. There are many interesting early-stage strategies, but if the goal is translational medicine, it is of utmost importance to take manufacturing issues into account from the very beginning. Of course, if you have such know-how, this challenge also presents an opportunity/entry barrier that can be capitalized on and should also present a significant barrier for generic competition. A scientist from Gilead told the story of the attempt by a fairly large Argentinian generics manufacturer to copy Gilead’s AmbiSome formulation by just mixing the individual components together and that this went quite wrong.

In order to address that gap between basic and applied science, the opening talk advocated the creation of companies whose sole business model is to take promising academic research and transform it into industrially partnerable technologies. Certainly, this concept appears to be gaining traction and it will be important in how the compete with or complement small biotech companies. The Centre for Drug Research and Development in Vancouver is one such entity that can be put into this category, also with a liposome focus (Pieter Culles e.g.). Vancouver in general can be considered the world's capital for liposomal research, which brings us to the Tekmira, UBC/Alcana, Alnylam triangle.

Pieter Cullis, co-founder of a number of liposomal companies including Tekmira, but now apparently more closely associated with Alcana and Alnylam, gave a nice talk about the rational design of lipids for use within SNALPs. Only small changes in the lipids can have large biological and physico-chemical effects. For example, by just modulating the chemical strength of the linkage of the fatty acids with the lipid head-group, SNALPs of very different potencies are obtained. Yet another objective is the investigation of the pKa-potency relationship which again can greatly impact liposomal performance. Through this, Alcana-UBC, largely driven by two ex-Tekmira scientists have come up with around 100 new lipids which further help to refine the design rules in an interative process. Actually, the rational lipid design approach is the approach Tekmira itself has been taking- probably not surprising given this background and may explain why Tekmira is getting access to these lipids for free without having to fund their development. It also differentiates it from the ‘lipidoid’ approach by MIT-Alnylam. Maybe given the varied performances due to even only subtle changes, huge libraries of lipids are not that desperately needed as was thought just 3-5 years ago. It is also the animal model that would become the bottle neck, as the tissue culture system is of only limited utility in predictively characterizing SNALP performance and mice become the system for the screen. Still, ‘lipidoids’ may offer fundamentally new structures which can be further refined using similar rational design approaches. For the liver, at least in terms of potency, this may not be needed according to Cullis’ back-of-the-envelope calculation that predicts single digit microgram(siRNA)/kg potencies that the technology is approaching for this tissue, an area that is approaching certain theoretical limits.

In summary, the conference, has confirmed my view that liposomal delivery is a good place to be in RNAi Therapeutics at the moment. It may even be time to have a conference entirely devoted to liposomal strategies for RNAi Therapeutics. There is a need, however, to focus on those companies that operate on sound scientific principles and ideally have a track record in being able to produce high-quality/consistent liposomes in a manner that is scalable.