Viruses, Beware! This time, RNAi Therapeutics Mean Business

Viral infections have long been thought of as an attractive therapeutic area for RNAi Therapeutics.  Unfortunately, with the exception of Tekmira’s Ebola biodefense effort funded by the US Department of Defense, this area has had trouble taking off: Nucleonic’s ddRNAi-based HBV program should never have gone into the clinic (and as expected was soon terminated thereafter), and there is considerable concern that the main mechanism of action of Alnylam’s ALN-RSV01 for respiratory viral infection is due to innate immune stimulation of the unmodified RNAi trigger, not RNAi-mediated gene knockdown.

As RNAi Therapeutics as a whole has turned the corner in 2012, so has antiviral RNAi Therapeutics. 

This assessment is based on two quality programs that have either made it into the clinic recently, Calimmune’s ddRNAi candidate for HIV (LVsh5/C46), or is close to it (Arrowhead’s DPC-delivered anti-HBV candidate ARC520 for which an IND is planned in Q2 2013).  In addition, there is expectation that Tekmira’s Ebola program will be able to take advantage of the significant improvements in SNALP delivery technology, thereby considerably increasing the odds for an FDA approval under the Animal Rule (note the recentapproval of a second drug under this rule).

Suppressing Immune Suppression

Antiviral RNAi Therapeutics have to overcome the important theoretical limitation that even a potent, e.g. 99% knockdown of a viral transcript or particles may not be sufficient as in theory a single infected cell may fuel viral rebound.  It turns out that rather than blindly aiming at knockdown potency, RNAi Therapeutics are likely to be more successful when targeting an important mechanism employed by virtually all viruses: avoiding detection or removal by the immune system.

In the case of HBV, a disease affecting North of 200 million patients worldwide, the virus produces large amounts of the Hepatitis B Surface antigen (HBsAg).  This is thought to suppress, by acting as a decoy, the development of a productive anti-HBsAg immune reponse.   It is thus widely believed in the industry that reducing HBsAg is required to finally generate a drug that can achieve a functional cure, essentially paralleling the recent developments in HCV.  Interferon-based treatment regimens may actually partially work via this mechanism, but cure rates are rather low and come with considerable side effects in the form of severe flu-like symptoms.  Moreover, protein-targeting anti-HBV agents such as small molecule-based polymerase inhibitors do not seem to reduce HBsAg.  This leaves RNAi Therapeutics as the most promising mechanism of action.

A recent article in PLOS Pathogen suggests that the Ebola virus similarly churns out decoy viral proteins so as to subvert the immune system into making antibody duds that do not effectively remove the real viral particles.  It is therefore intriguing that an Ebola drug candidate by Tekmira should not only aim at providing the immune system with more time, but also that it would facilitate it mount a more effective antibody response.

No Escape

Another attraction of the RNAi Therapeutics approach for viral diseases is the fact that such agents may be more successful in prohibiting the virus to mutate around the drug and thereby escape its actions (viral escape).  Consequently, all antiviral RNAi trigger selection strategies focus on sites that are conserved in the various genotypes and quasispecies.  Even if the virus is successful at mutating around conserved sites, it is then relatively simple to include a second (such as in Tekmira’s Ebola program) or third RNAi trigger targeting a conserved site such that the virus would have to mutate around two sites at the same time- a highly unlikely event.

In addition to these general antiviral mechanisms, RNAi Therapeutics may also work through more virus-specific mechanisms.  Calimmune’s ddRNAi-based HIV candidate LVsh5/C46 for example down-regulates the cellular receptor for viral entry, CCR5, such that HIV particles cannot enter cells and integrate into their genomes in the first place.  As an ddRNAi gene therapy approach, LVsh5/C46 further takes advantage of the fact that you can express a therapeutic protein along with the RNAi trigger, thus uniquely combining mechanisms of actions in a single drug.    

Smooth Sailing Ahead

Of course, it is impossible to tell whether an RNAi Therapeutic will actually overcome a virus in each case and receive regulatory approval.  Nevertheless, I believe that the above candidates for Ebola, HBV, and HIV stand a real chance. 

The Ebola program by Tekmira is arguably the most advanced, and it is difficult for me to see how based on the non-human primate data and the lower dosages required for SNALP delivery, which should widen the therapeutic window, approval can be denied under the Animal Rule.

For the HBV and HIV candidates that are being developed along more conventional regulatory pathways, I  am similarly optimistic that they will generate some excitement in the near-term.  This is because viral load is a powerful biomarker, often also an approvable endpoint, and even early clinical studies should be able to generate such outcome data (if Arrowhead could help it, they should go straight into patients with ARC520). 

After orphan diseases involving the liver and oncology, antiviral applications are therefore poised to become the third major support of the RNAi Therapeutics platform.

Solid Calimmune DNA-directed RNAi Therapeutics Candidate for HIV Nearing Clinical Development

With the backing of a $20M grant from the California Institute of Regenerative Medicines (CIRM), Calimmune has made progress in advancing a DNA-directed RNAi (ddRNAi) Therapeutics candidate for the treatment of HIV/AIDS towards clinical development in early 2012 (here a recent blurb in the Financial Times). Similar to an HIV candidate developed by City of Hope (CoH) and Benitec before it, the new treatment involves the modification of a patient’s own blood stem cells (hematopoietic stem cells, HSC) with a gene therapy comprising of an expressed small hairpin RNAi trigger. Although Calimmune is not prepared yet to share the details of this program, based on my review of the research conducted by groups associated with Calimmune, the likely candidate has the potential to become one of the most exciting ddRNAi Therapeutics product candidates to enter the clinic yet.

HIV therapy today and motivation for gene-based stem cell therapies

The treatment of HIV has made tremendous progress. Once a certain death sentence, for those with access it has instead largely become a chronic infection that can be kept in check with cocktails of small molecules targeting a variety of stages in the viral life-cycle (highly active antiretroviral therapies or hAART). Nevertheless, the need for taking daily pills for life comes at the cost of side effects, generally reduced quality of life, and the emergence of viral resistances. There is no cure yet for HIVAIDS.

Actually, there might be one example of a cure for HIV. In 2006, an AIDS leukemia patient, aka the Berlin patient, underwent a bone marrow transplant as a treatment for his leukemia. The doctors selected a bone marrow donor whose cells carried defects in the CCR5 gene on both chromosomes. After the transplantation, the patient was not immediately put back on antiretroviral therapy to allow for recovery of his new hematopoietic system. Surprisingly, despite the absence of drug treatment, the virus has not recurred to this day leading more and more experts to talk of the first functional cure of HIV/AIDS.

In hindsight, this result did not come totally as a surprise. CCR5 had been known to be an important entry receptor for the common CCR5-tropic HIV isolates. Epidemiologic evidence gathered in the mid 90s indicated that people with certain CCR5 deletions on both chromosomes were protected from HIV infection, and those with a CCR5 defect on only one chromosome had, on average, delayed disease progression and improved life expectancies. In fact, this research led to the development and recent approval of a class of drugs blocking the CCR5 protein (e.g. Maraviroc by Pfizer).

There remains, however, great interest in developing gene-based stem cell medicines against CCR5 (and other HIV viral and host targets) in the hope of generating HIV medicines with less side effects, reduced chance of viral resistance (one way of HIV resistance to drugs targeting the CCR5 protein is to bind to CCR5 in the presence of drug), and maybe even a cure. The Berlin patient indicates that CCR5 may be an ideal target for such gene-based stem cell therapies.

Two possible mechanisms by which such a strategy may succeed are based on eradication of HIV-permissive cells as they are killed off by the virus while the CCR5-impaired cells persist, or by improving the immune function of CCR5-impaired cells thereby allowing them to fight HIV infection in other places.

City of Hope/Benitec and the first DNA-directed RNAi Therapeutic for HIV

Calimmune’s ddRNAi candidate is not the first one for HIV. The City of Hope, with the financial backing of Benitec, already entered one into clinical development (rHIV-shl-TAR-CCR5RZ), results from which were reported last year in Science Translational Medicine. Recognizing the advantages, if not need, for targeting multiple stages of the HIV life-cycle at once, this candidate was not a pure ddRNAi therapeutics, but a triple RNA therapeutic that in addition to the shRNA RNAi trigger which targeted the viral tat/rev mRNA involved an expressed TAR RNA decoy and an expressed (RNA) ribozyme targeting CCR5. Notably, all three expression cassettes were driven by U6 promoters.

The expression cassettes were placed in a shared lentiviral vector and thus introduced ex vivo, i.e. outside the body, into hematopoietic stem cells isolated from the enrolled AIDS lymphoma patients. Because hematopoietic stem cell transplantation with full bone marrow ablation is associated with risks, but is standard second-line therapy for AIDS-related lymphoma, this patient population was chosen so that the trial participants would simultaneously receive a treatment benefit for their lymphoma while participating in this experimental trial. As an added measure of precaution, the majority of hematopoietic stem cells were left untreated and given together with the modified stem cells to ensure that the immune system would be reconstituted even if something went wrong with the gene therapy.

Four patients were treated per protocol in the phase I trial. Unfortunately, while there was no obvious significant adverse event as a result of the gene therapy, the molecular analyses indicated that rHIV-shl-TAR-CCR5RZ may not be the most promising RNA therapeutics candidate for HIV. Specifically, while the initial transduction efficiency was in line with what would have been expected for lentiviral delivery (~20%, see X-linked adrenoleukodystrophy trial here), the transduced cell population declined rapidly and the ones that persisted were just about detectable- too few to be therapeutically promising.

If this candidate were to be further developed, an important goal would be to increase the fraction of stem cells that are modified. This could either be by improving the transduction efficiency, by only providing stem cells that were treated with lentivirus instead of providing the untreated stem cells as a backup, or by using a protocol that chemically selects for the modified stem cells after their re-infusion. Still, I am skeptical that this would solve the problem as in light of other lentiviral and retroviral clinical experiences the observed decline in transduced cells seemed to be specific to rHIV-shl-TAR-CCR5RZ. It is therefore possible that some inherent toxicity of the expression cassette itself, possibly due to the use of U6 promoters, accounted for the poor long-term persistence of modified stem cells.

The Calimmune approach: A non-toxic, H1-driven shRNA targeting CCR5

The reason why I feel that Calimmune’s approach may have better prospects is that it has fully accounted for the U6-related shRNA toxicities and selected an H1 promoter-based RNAi expression cassette that was shown to be both safe/stable and, equally important, highly efficient in CCR5 knockdown in human and rhesus HSC-derived cells. Also, I like the fact that it is an RNAi trigger, and not a ribozyme, that is targeting CCR5, as I believe this to be the more efficient knockdown modality.

While Calimmune has yet to fully disclose their eventual clinical candidate, the one reservation that I have about the putative candidate at this time is that they may have failed to take advantage of the combinatorial potential of RNAi Therapeutics. With combinatorial potential I do not necessarily mean here combining ddRNAi with other RNA (like CoH/Benitec) or protein expression modalities- in fact, it may be scientifically 'cleaner' to use just RNAi for now- but targeting at least two HIV-related genes instead of one to minimize the emergence of viral resistance.

The panels on the left depict what in my mind have been the most impressive dataset from the development program. It shows the results from a rhesus monkey model in which the ddRNAi trigger was introduced into blood stem cells from two monkeys (RQ3570 and RQ5427 for those with good eyes) which (panel A) led to solid, long-term (!) 6-20% cell marking in the various cell lineages of the blood. Moreover, when the cells were sorted into those that were transduced (black bars, panel B) versus those that were not (grey bars, panel B) and the CCR5 levels measured in the respective cell populations, the CCR5 was found to be down-regulated by 80-90% in the transduced cells. And since your experiment is only as good as your negative controls, data from a control animal that received a lentivirus without the RNAi trigger (2RC003) show no differences in CCR5 levels between the two cell populations.

While I have yet to see the obligatory HIV in vivo challenge studies with this putative candidate, based on CCR5 genetics, a candidate with such transduction levels and knockdown potencies should stand a good chance at improving CD4+ T-cell counts for enhanced immune system vigor and delaying or maybe even eradicating HIV over time.

It is debatable to what degree a full CCR5 knockout compared to a highly potent CCR5 knockdown would bring additional benefits. Sangamo Biosciences for example has made tremendous progress in increasing the efficiency of gene disruption using their Zinc Finger Nuclease technology. Not surprisingly, this company also has a CCR5 hematopoietic stem cell candidate in the early pipeline. In a 2010 Nature Biotechnology paper, Sangamo reported an estimated frequency of 5-7% homozygous CCR5 gene disruption in human hematopoietic stem cells, and another 10% heterozygous gene disruptions.

Simplistically, taking upper estimates, ddRNAi may provide for 90% CCR5 knockdown in 20% of cells whereas ZFN technology may delete CCR5 altogether in 7% of cells and knockdown CCR5 by half in another 10%. Because these numbers are close and a clean knockout in some cells may make up for the slightly decreased overall knockdown levels, I would be even more excited to see Calimmune's current lead candidate paired with at least another shRNAi trigger, thereby exploiting said combinatorial potential of ddRNAi Therapeutics which ZFNs cannot provide as easily.

Benitec license?

Benitec, of course, will follow Calimmune’s developments with great interest as the company has rights to critical ddRNAi trigger patents. Curiously, both companies are based in Australia, but have significant roots also in the US South-West, so it should be possible to come to an amicable agreement.

License or not, it will be good for the entire field of RNAi Therapeutics, and ddRNAi Therapeutics in particular, for this trial to get underway in 2012 as it should attract significant general interest to a what looks like a solid RNAi Therapeutics candidate.

Acknowledgement: The idea for this blog came from a reader that alerted me to this interesting RNAi Therapeutics candidate that had flown below my radar, and maybe also to placate another reader that complained that the Tekmira-Alnylam feud was taking up too much space and there were other interesting things happening, especially in ddRNAi Therapeutics. So if you know of exciting RNAi Therapeutics developments that you believe I may be missing, please let me know by email (first name dot last name at gmail dot com). In most cases, I won’t be able to write about it immediately, but it won’t be forgotten either.

Update: On March 5, 2012, Calimmune acquired a global, non-exclusive license from Benitec to use ddRNAi in HIV/AIDS.

Nucleonics in Liquidation While Benitec Shows Signs of Life

Just a day after boasting how RNAi Therapeutics continues to enjoy ample financial support in a tough economic environment, a google news alert indicates that Nucleonics is in liquidation mode.

Since its inception, Nucleonics has made more headlines with their IP battles with Benitec over DNA-directed RNAi supremacy rather than with good science, and must be filed under those early biotech companies that spent more money on administrative and legal expenses than R&D. The ultimate nail in the coffin may have occurred when a Federal Court denied Nucleonic’s wish for a declaratory judgment against Benitec’s patent claims last year, thus leaving the company and investors vulnerable to future lawsuits by Benitec.

While it has to be said in Nucleonics's defense that it wasn't solely responsible for the endless litigation, the obvious winner from this new development is Benitec. With their arch rival out of the game it may now find it easier to concentrate on drug development, although financing and IP issues remain of concern. Encouragingly, their collaborator on the HIV AIDS lymphoma program, John Zaia from the City of Hope, just recently presented at the annual ASGT meeting early interim phase I data on the successful transplantation of RNAi-modified hematopoietic stem cells in two patients (using lentiviral vector technology). It will be exciting to determine how safe and sustained RNAi expression is and whether the derived T-cells have a survival advantage compared to those derived from the unmodified stem cell fraction transplanted at the same time.

Coming back to Nucleonics, the apparent bankruptcy is also likely to affect their recent HBV RNAi clinical program involving the administration of a plasmid formulated with cationic lipid. We may never know about what happened to the first patients that received the plasmid, and maybe that’s good so. The Nucleonics experience shows that early IP battles are dangerous and costly, and that as the RNAi Therapeutics field matures it is becoming difficult to attract funding based on me-too technologies and long-shot scientific strategies.

Disclaimer: This Blog may not be based on reality and reflects my views as of today only.

RNAi Therapeutics: Qiagen High-Throughput RNAi User Meeting San Francisco (Part 2 of 2)

After the coffee break, John Hogenesch illustrated how RNAi has revolutionized our concept of mammalian genetics. Before RNAi, researchers essentially studied two expression states of a gene: when it was fully expressed (+/+), or when both alleles had either lost their function or weren’t expressed at all (-/-), and rarely the heterozygous state (+/-). RNAi, however, now allows the functional output of a gene to be measured in virtually all the states in between, depending on the RNAi dosage used.

Hogenesch’s systems biology team is interested in how the circadian rhythm is genetically wired that that it can be robust and yet adaptable, and consequently set up a beautiful system whereby the activity of master transcription factors of this genetic program could be measured in real-time over days based on the luminescence generated by binding of the transcription factors to a reporter gene. They then knocked down known regulators of this genetic circuit to varying degrees by titrating the siRNA amount and then assessed the correlation between the degrees of knock down and functional outputs. Pleasantly surprising not only Hogenesch, but also all the researchers worrying about how the degree of knockdown affects the phenotype they are seeing in RNAi experiments, in many cases the functional output appeared to almost directly correlate with the remaining expression level of a gene after knockdown.

However, in cases where there exist paralogues, that is highly related genes that have largely retained overlapping functions, the knockdown of one gene is often compensated almost entirely by the increased expression of the corresponding paralogue. You probably may want to avoid targeting these genes with an RNAi Therapeutics, or any other therapeutic for that matter. Finally, there were also cases where the knockdown gave non-linear responses, and this is mostly when enzymatic reactions were affected.

Nevertheless, as everybody in the audience having traveled from across the country for this one-day meeting knew, genetic circuits, long-term, are usually quite robust and can adjust to external fluctuations. It is therefore also of interest to RNAi Therapeutics (and then again, all classes of drugs, too), particularly for chronic applications, that out of the ~4400 gene knockouts in mice that have been generated so far, only 1000 are lethal in the homozygous state, and just 53 in their heterozygous state, and these already are probably biased numbers (scientists like to study genes with apparent phenotypes, lethality being a very obvious one). This has implications both for chronic drug treatment in general (drug effect may diminish) as well as when we worry about adverse consequences of off-targeting (off-targeting may be tolerated better than expected).

Next up was Loren Miraglia from the Genomics Institute of the Novartis Research Foundation (GNF, San Diego), a basic research institute with preclinical capabilities to feed into the Novartis drug development pipeline. The main part of the presentation focused on the technical aspects of establishing a lentiviral RNAi library for the investigation of difficult-to-transfect cells. The upshot was that these libraries once established can be very powerful, however it takes a considerable infrastructure to realize that goal.

There were two pieces of information that may give some quite interesting insights into Novartis’ RNAi Therapeutics plans. One was an overview of the RNAi libraries being used by GNF which essentially all somewhat disappointingly were focused on the “druggable genome”, disappointing because a lot of genes may be missed that could be addressed by RNAi Therapeutics. There was one notable exception, however, and that was a library devoted to genes involved in cancer. As the time approaches for Novartis to make their move on Alnylam, this confirms my belief that cancer will be an important part of the new license agreement. With screening efforts such as these, there should be more than enough targets to keep all the Alnylam licensees busy without having them compete too much with each other.

The second insight was Miraglia’s presentation of a new “gene X” that when mutated had been found to lead to an increase in LDL-R, the new star in the hypercholesterolemia field. When tested whether RNAi knockdown would also elevate LDL-R, this was indeed the case with a nice correlation between degree of target knockdown and LDL-R elevation and subsequent cholesterol lowering, just what you would like to see in an RNAi Therapeutic. So, like for Takeda, cancer may be well joined by metabolic disease as one of the main initial therapeutic fields for Novartis’ RNAi Therapeutics efforts and driven by current delivery capabilities.

Systems biologist Sumit Chanda (Burnham Insititute, La Jolla) praised RNAi screening as an important tool to learn more about the vast part of the genome that is essentially left unexplored. He bemoaned the fact that it is paradoxically genes of which so much is already known about, think of p53 and TNF-alpha, that get the bulk of the research funding.

Chanda’s laboratory used RNAi screening to discover host factors involved in HIV replication, which could therefore also be potential targets for drug intervention. As a virologist/molecular biologist myself, I also see targeting host factors as a very promising approach for treating viral infection. Unfortunately for Chanda’s group, another group from Harvard just published the results of a similar RNAi library screen, meaning that they were “scooped”. Nevertheless, it appeared that there was only limited overlap between the results and one could explain this by the slightly different assay conditions employed or the quality of each screen. In any case, the point was well made that one screen will never answer all the questions and should be complemented by additional ones. Moreover, the importance for multiple negative controls and multiple redundant siRNAs towards a single gene (to confirm the sequence-specificity of the hit) was emphasized.

Natasha Caplen from the National Cancer Institute (NCI; Rockville, MD) picked up the personalized medicine theme for cancer therapy. As noted before, while monoclonal antibodies have changed the way cancer is treated today, it is often only few patients that respond to them. Moreover, many of the drugs’ mechanism of action converge on the few same signaling pathways, meaning that the mechanisms of actions are still limited and prone to mutational escape. RNAi Therapeutics could then either play a role in exploiting new mechanisms of actions or for sensitizing towards existing drugs. Caplen’s group chose the latter approach and focused on devising new strategies for the use of the bacterial L-Asparaginase enzyme (L-ASP) that has been used to selectively starve acute lymphoblastic leukemia (ALL) cells. One problem associated with L-ASP, however, has been the quite variable response to this treatment.

Caplen’s group suspected that cancers that do not respond well to L-ASP treatment may overproduce the human asparagine synthetase (ASNS) to compensate for asparagine deprivation by L-ASP. To test their hypothesis they first screened a library of cancer cell lines for L-ASP responsiveness and, indeed, L-ASP negatively correlated with ASNS expression levels. To demonstrate a causal relationship, they then knocked down ASNS which greatly increased the sensitivity of the cells to L-ASP. The sensitization was so unbelievable, up to 500-fold, that Natasha Caplen made her co-worker repeat the experiment again and again to convince her of the veracity of the results.

ASNS expression levels could now be used to select patients for L-ASP treatment. Alternatively, ASNS knockdown may be a viable way to increase L-ASP responsiveness. It more and more appears therefore that cancer drug sensitization, and not just targeting novel oncogenes appears to be a very promising avenue for future cancer RNAi Therapeutics. Similar patient selection and drug sensitization strategies were subsequently discussed for the topoisomerase inhibitor camptothecin (Caplen) and the Wnt signaling pathway by Paul Kassner (Amgen).

Finally, I’d like to summarize the talk given by Xiao-Dong Yang from Intradigm (Palo Alto, CA) on the delivery of RNAi Therapeutics to cancer. Using siRNAs provided by Qiagen, it appears that Intradigm screens for siRNAs essentially based on potency. I am somewhat skeptical whether this is sufficient and consequently raises some questions as to their findings that were largely centered on targeting the VEGF pathway in mouse cancer models.

Intradigm’s RNAi Nanoplexes consist of a cationic polymer to which the siRNA is complexed, a hydrophilic steric polymer such as PEG, and optionally a cell targeting ligand such as an RGD peptide. The Polytran polymer-based system is particularly interesting as it is a polypeptide composed of branched histidine and lysine residues. While the positively charged lysine condenses the siRNA, histidine only becomes positively charged once internalized into the endosome as they suck up the incoming protons. This supposedly leads to an increase in osmotic pressure causing the rupture of the endosomes to release the siRNA into the cytoplasm.

The Polytran system consists of fairly uniform 100nm particles that are slightly positively charged. Like many RNAi nanoparticle systems, of the Nanoplexes that make it into a tissue following administration, many are either found in the liver, the spleen, or tumor tissue. Many of them are also taken up by phagocytic cells such as macrophages which may pose challenges with dose prediction and requires that siRNAs are carefully characterized as to their potential to activate the innate immune system. Unfortunately, this aspect of their work was little discussed although unmodified siRNAs, prone to elicit innate immune responses and could therefore cause non-specific anti-tumorigenic effects, were used. With this in mind and the fact that only one control siRNA sequence was used, quite potent mouse antitumor effects were found at low mg/kg dosages with apparently knockdown efficiencies of up to over 90%.

While these approaches are valid for the development of an RNAi delivery system, the presentation reminded me of the fact that for companies like Intradigm it would really make sense to collaborate on the siRNA chemistry side with one of the established RNAi operations rather than committing insufficiently characterized siRNAs into the clinic.

On the way home, I was once again amazed by the fact that RNAi has emerged as the only gene knockdown technology that can be applied in high-throughput to explore the depths of our genomes. This both speaks to the potency and specificity of RNAi as well as the ability to translate our understanding of how RNAi silencing works on a molecular level into bioinformatic tools to separate the wheat from the chaff, as Sumit Chanda put it.

Thanks a lot to Qiagen for organizing a day full of excellent scientific talks in a pleasant Mission Bay setting, and I’d recommend everybody running qPCRs Qiagen’s robust and reliable SYBR Green QuantiTect reagents.

Day 4 of the RNAi Keystone Conference

As I alluded to already in my previous post on the conference, the issue of transcriptional gene silencing (TGS) mediated by RNAi-related mechanisms in mammals has been somewhat clouded. If it existed and the rules for inducing them could be clearly laid out, it may represent an alternative means of harnessing RNAi for therapy. Presentations on the (at least in mammals) germline-specific Piwi-associated small RNAs (piRNAs; by Alexei Aravin, Cold Spring Harbor) in mice and the small RNA profile from human embryonic stem cells showing that many of them correspond to transposable elements, often regarded as the parasites of our genomes and therefore have to be contained (e.g. by RNAi), the nuclear localizations of Dicer (Witold Filipowicz, Basel) and Argonautes (poster by the Meister group in Munich), and various data on the nuclear localization, and possibly processing, of microRNA precursors into mature microRNAs, are all very suggestive that promoter-targeted TGS may be part of biologically-relevant gene regulation and have promise as a therapeutic technology platform.

In addition to the nuclear localization of Dicer, there was additional data on this central RNAi enzyme. What is often so amazing about science and was also demonstrated a number of times at this conference, different groups working on apparently different phenomena converge on the same results. John Rossi (City of Hope) for example had been studying whether DNA-directed long shRNAs could be processed such that HIV would be targeted by more than a single small RNA, thereby minimizing the likelihood of escape mutants. Like the structural biologist Jennifer Doudna (Berkeley), he finds that mutations in the helicase domain converts human Dicer from an enzyme that does not like to processively chop long dsRNAs into a string of short RNAs into one that does it with reasonable efficacy. Of course, we always hope here that findings like these may allow us to adjust therapeutic designs such that in this case long DNA-directed shRNAs may be processed more efficiently by Dicer. From a basic biology perspective, studies such as Doudna’s on the structural requirements of Dicer should also encourage us to consider endogenous RNAs as Dicer substrates even if they do not result in small RNAs.

Somewhat surprising to me, Thomas Tuschl’s (Rockefeller) presentation focused on the use of microRNAs as diagnostics. It is worth remembering that his group discovered and patented many of the first human microRNAs and that IP may have most immediate value in diagnostics. Specifically, his group is refining technologies for detecting microRNAs in situ (using LNA-spiked probes) and the quantitative profiling of microRNAs by counting. It appears to me that while first-generation microRNA diagnostics in the clinic will involve interrogating a limited number of microRNAs by qRT-PCR (one of Rosetta’s first diagnostics for squamous versus non-squamous lung cancer appears to be based on the PCR quantitation of a single microRNA), while in another 10-20 years quantitative sequencing technologies take over, possibly by-passing array-based detection methods for clinical use altogether.

Phil Sharp (MIT) reminded us that the complexity of the transcriptome may have important implications for our understanding of microRNA regulation. 3’ UTRs are the main site of microRNA action (although there were some bioinformatics talks at the meeting that strongly supported functional microRNA target sites in the open-reading frames of genes as well, though to a much lesser extent than in 3’ UTRs) and may be dynamically regulated according to the state of the cell, e.g. proliferation. Since the extent of these 3’ UTR changes (due to alternative splicing or polyadenylation) may be quite considerable, this complicates efforts to unravel the functional microRNA interactions for the identification, including safety, of candidate microRNA therapeutics.

High-throughput RNAi library screening for drug target identification (commercial examples are Cenix Biosciences, Galapagos Genomics), using synthetic siRNAs as well as plasmid and viral-based shRNA, is now well established. According to Ed Harlow (Harvard), this really has opened up mammalian biology for the type of genetic experimentation long reserved to model organisms. Focusing on the human kinome and cancer (all the predicted human kinases), he made the observation that the functional outcomes of suppressing a given gene may differ considerably according to the experimental system (e.g. depending on the cell lines). This should serve to caution us that context such as the presence of an oncogene or tumor-suppressor gene or not and redundancy can have a profound impact on the importance of a kinase in cell signaling. Another observation from his screens was that the resulting drug target candidates coming out of these screens were not biased towards the kinases that have been already subject of the majority of kinase-related studies, illustrating how RNAi may also benefit small molecule drug development. While in vitro RNAi screens are common, the costs associated with high-throughput in vivo screens would seem prohibitive. It is therefore notable that Michael Hemann’s (MIT) approach to studying the role of genes in cancer drug resistance involves the transduction of a library of shRNAs into cancer cells of the blood and administering them to mice. Changes in the relative abundance of a given shRNA prior to and after drug treatment are then monitored as a measure of the importance of the targeted gene in drug response and resistance. I could imagine that similar approaches to extend the power of RNAi screens may have near-term potential not only for cancer, but also other settings where it is possible to select for relative cell viability (viral infection, degenerative diseases etc).

John Rossi (City of Hope) reported on progress with the first DNA-directed RNAi gene therapy trial (corporate sponsor: Benitec) involving a triple RNA therapeutic against HIV, including a U6-driven shRNA, lentivirally delivered to bone marrow transplant cells in AIDS lymphoma patients that are in need of a BMT anyway. This is a limited trial with an ultimate enrolment goal of 6 patients. Given the complexity of the therapeutic and trial designs it has taken considerable efforts before the first patient could be dosed, but happily the first patient has now been treated a couple of weeks ago. It will be exciting to follow the progress of this patient. Since he/she also received untreated BMT cells for safety reasons, it should be possible to monitor the enrichment/non-enrichment of treated vs untreated T-cells as an indicator of treatment success. Since lentiviral vectors have not been associated with oncogene activation, probably my main safety concern for this trial is the use of a U6-driven shRNA. Not only did having two U6 promoters in one vector cause excision of the U6-shRNA cassette due to a recombination event in some of the genomically integrated vectors, it is known that U6-shRNAs have a tendency to be toxic due to sheer promoter strength as well as possibly its particular processing. For future DNA-directed RNAi development programs, the use of H1 promoters may therefore be even more promising.

A word here on viral escape mutants to RNAi Therapeutics as antivirals. The triple construct in the HIV trial was based on the notion that hitting the virus through three different mechanisms should limit viral escape. As such, it was reported in studies leading up to this trial that HIV likes to mutate around shRNA target sites. To my knowledge, this had not been reported for the HIV sequences targeted by the ribozyme and decoy components in this vector. Contrary to popular opinion, I am actually quite pleased to see such mutations being selected for since this is a clear indication of the efficacy of the shRNA. Moreover, with RNAi the target site for the shRNA could be chosen such that largely unfit viruses were selected for.

Although all three components of the triple construct are RNA-based therapeutics, it illustrates the potential of combining RNAi Therapeutics with other mechanisms of actions into one drug. Similarly, Rossi’s group is now developing an aptamer designed to both neutralize circulating HIV virions as well as target attached anti-HIV siRNAs to HIV infected cells. Another example may be liposomally delivered siRNAs, which in the case of a cancer therapy could be designed such that not an oncogene would be targeted, but also such that the RNA would stimulate the immune response against the cancer. Interestingly, a poster presented by Nigel McMillan (Brisbane, Australia) reported on the maybe somewhat surprising finding that the targeting of an mRNA led to the production of a truncated protein (presumably by translation from the cleaved mRNA) which stimulated an immune response against the oncogene with, if it holds up, some interesting implications for microRNA evolution (cleavage vs non-cleavage) and RNAi Therapeutics.

The aptamer-siRNA combination involved the Dicer-substrate design. The rationale being that such a combination necessitates the covalent attachment of the RNAi trigger to the carrier so that Dicer cleavage has to liberate the active small RNA from the carrier. This is reasonable, but one should note that smaller siRNAs covalently attached to cholesterol on the passenger strand, and probably also the 3’ end of the guide, are also known to be bioactive (although I am not sure whether some efficacy was compromised by such designs). Also, I would be curious to see whether attachment via disulfide bonds that would break in the reductive cytoplasm would liberate even larger amounts of bioactive dsRNAs, both long and small, thus further illustrating that covalent attachment to the delivery vehicle does not represent a fundamental disadvantage for the delivery of short siRNAs.

Of course, this gets us right into the IP issue. A poster by RXi was instructive of how companies aim to get around Alnylam’s perceived dominance in this arena (almost like watching a virus mutate around an shRNA target site). As many of you know already, the focus is on the use of dsRNAs longer than 23bp as well as blunt-end versus overhang dsRNAs. Without discussing the relative scientific merits of the different approaches, it is notable that RXi’s poster put great emphasis that their 25bp Stealth siRNAs were not processed by Dicer into smaller siRNAs as would have been expected for equally long unmodified dsRNAs, yet were still active in gene silencing. Unlike Dicer-substrate oriented approaches, RXi’s intention is to circumvent any claims by Alnylam that Dicer-substrates are nothing more than pro-drugs to the classical Tuschl design, certainly novel and non-obvious from an argumentation point of view. Scientifically, I would be interested in whether these dsRNAs work as efficiently in Dicer knockdown or knockout cells, in other words whether Dicer would still recognize these particular dsRNAs and facilitate RNAi by handing them over to RiSC, independent of whether Dicer had processed or not, and whether this rather than dicing is how Dicer facilitates gene silencing for a range of non-diceable RNAi triggers. In other words, since Dicer is known to aid in loading the RNAi effector complex, RiSC, aren’t all dsRNAs, including siRNAs, Dicer substrates and would the size of microRNAs/siRNAs be a consequence of Dicer processing, but not necessarily a requirement for RNAi activity? Tuschl’s early work on single-stranded RNAi, showing that ssRNAs of various length, i.e. not only 19-21mers, but also 29mers and others, were equally efficient in triggering RNAi suggests just that [Martinez et al. (2002). Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 110:563].

Local Tekmira had a poster on the 10-fold improvement in efficacy of some of the new liposomal nanoparticles down to 0.1mg/kg, similar to what had been reported on Protiva's website and at Alnylam's R&D day. But rather than discussing here more about SNALP-like technology which I have done extensively in the past, and before I will summarize the conference’s last day, I will have to first read up on the Tekmira news that came out today, an apparent victory of reason and impressive management skill.

Benitec and City of Hope Start RNA-targeted HIV Phase I Clinical Trial

After some delay, Benitec and their collaborators from the City of Hope finally announced that they obtained regulatory approval to start phase I clinical trials for an RNA-based HIV gene therapy antiviral. The delay was caused by Benitec’s uncertain corporate future and difficulties generating sufficient amounts of clinical-grade lentiviral vectors, but scientifically the risk-(potential) benefit profile of these studies are promising.

This is the 8th RNAi clinical program and the second involving DNA-directed shRNAs. The gene therapy agent is a lentiviral vector expressing 3 different RNA molecules, each designed to interfere with a different aspect of HIV replication, each through a distinct mechanism of action. The plan is to immunise CD34+ hematopoietic progenitor cells and thereby all their progenies, including T-cells, with such vectors ex vivo and then re-administer the cells to the patients.

The RNAi portion is a U6-driven hairpin RNA targeting the tat/rev mRNA by RNAi. A nucleolar-localised TAR decoy RNA, also under the direction of a U6 promoter, is designed to mimic the HIV TAR element, thereby diverting TAR-binding factors by mimicry. Finally, a ribozyme against the CCR5 mRNA, a co-receptor for HIV infection, complements the 3-pronged approach. This approach is inspired by the current HAART HIV treatment paradigm where a cocktail of antiretroviral drugs has proven to be highly effective in suppressing HIV replication with only slow development of drug resistance.

In fact, the RNA-based vector which has been shown in tissue-culture experiments to be considerably active in inhibiting HIV replication may be synergistic with present therapies due to their unique mechanism of action, although non RNA-based anti-CCR5 treatments are currently developed by other companies as well. While Benitec used to pursue a triple RNAi approach for the treatment of HCV (a program now owned by Tacere), the present strategy may be advantageous since it is known that co-transcribed shRNAs or co-transfected siRNAs may compete with each other for cellular RNAi factors.

Ultimately, I expect that long-term expression of ideally all 3 RNAs will be important to confer a survival advantage onto the lentivirally transduced CD34+-derived cells. However, even if expression should be silenced eventually, the treatment is likely to give AIDS patients some reprieve. The study population will be 5 AIDS-related leukaemia patients from which CD34+ will be enriched from blood by apheresis, genetically modified, and then returned to the donor patient.

Given that this is a gene therapy with a vector that hasn’t been used in the clinic before, it is expected that this therapy will be used in AIDS/Lymphoma patients who are no more responsive to conventional treatments. Nevertheless, expect to hear results from this promising phase I trial within a year.