Impressions from the Abstracts of the 7th Annual Meeting of the Oligonucleotide Therapeutics Society (Part 1)

The Annual Meetings of the Oligonucleotide Therapeutics Society are among the best on the conference circus related to, well, oligonucleotide therapeutics drug development. One benefit of bringing together RNAi Therapeutics, traditional RNaseH and steric block antisense, aptamers, and a few other oligo-based approaches is that researchers can benefit from sharing lessons in safety, how pharmacology relates to chemistry and formulation, manufacturing etc. Remember, it is the experience with older oligonucleotide technologies that allowed RNAi Therapeutics to take 10, instead of 20 or 30 years, to get to where it is today: over a 1000 patients and healthy volunteers dosed with more than a dozen of RNAi candidates exhibiting a decent, and improving safety profile; the ongoing Atu027 and ALN-TTR01 trials having reached dose levels where, based on sound science, robust target gene knockdown, technologically the primary objective, can be expected. Moreover, data from hypercholesterolemia, solid cancer, ocular and respiratory disease studies have provided evidence of dose-related therapeutic efficacy.

Not able to attend the 7^th Annual Meeting to be held this week in Denmark myself, I eagerly went through the abstract book to learn of new developments and trends. Here are my thoughts on a few select abstracts that I thought might be of interest to the readers of this blog (presented in the order they appear in the book). Note that if you are a Tekmira investor, keep reading until the end. Part 2 of the discussion can be found here.

Oral presentation: Expanding the structural diversity repertoire of siRNAs (Dong-Ki Lee, Sunkyunkwan University, Korea)

This presentation highlights the realization that a number of non-Tuschl RNAi trigger structures are not just IP workarounds, but can be used to achieve novel biological outcomes such as targeting multiple genes with one RNAi trigger molecule (multipodal structures), inducing select innate immune stimulation while at the same time silencing genes (long siRNAs), and reducing off-targeting (asymmetric siRNAs and ‘wobbly’ siRNAs).

Oral presentation: Activation of RNA interference in animals with single-stranded oligonucleotides (Erice Swayze, ISIS Pharmaceuticals)

For some indications, the intravenous application of the nanoparticle RNAi formulations which are leading in terms of in vivo RNAi gene silencing potency may be a commercial drawback (for the purpose of long market exclusivities, I believe it is a widely underappreciated benefit). ISIS Pharmaceuticals, until recently in collaboration with Alnylam have been working on naked single-strand RNAi (ssRNAi) solutions that can be administered subcutaneously.

It has been long known that ssRNAs can induce RNAi gene silencing, just 100-1000 less efficiently, which is not surprising since RNAi has evolved as a dsRNA-induced mechanism. The abstract claims that using fully modified, partially phosphorothioated ssRNAs, they were able to come within 5-fold of the potency of corresponding double-stranded structures. The initial animal experiments, however, seem to have failed due to ssRNA instability, but after further modifications they have now achieved activity at ‘pharmacologically relevant doses with subcutaneous administration in saline formulations’.

Certainly an interesting abstract and it remains to be seen just how pharmacologically relevant these doses are and the related safety profile. Similar, or better to their current RNaseH antisense? ssRNAi...ISIS’ antisense 3.0? Another interesting question is at what point did Alnylam drop the ball on ssRNAi after considerable investments- before or after the initial animal experiment failures?

Oral presentation: Delivery of Nucleic Acids (Muthiah Manoharan, Alnylam)

Alnylam’s oral presentation will be, you already guessed, about RNAi delivery. By listing 25 papers on two pages without any meaningful comment or discrimination, the abstract obviously wants to make the point that Alnylam is the leader also in RNAi delivery. Somewhat reminiscent of Alnylam’s press releases that used to list seemingly all their RNAi trigger-related patents, no matter how relevant to their gate-keeping potential which was the reason for listing them in the first place. As such, the abstract carries the dubious distinction of being the longest one of the conference, but the one with arguably the least content.

It is not the amount of money spent, the numbers of patents (‘thousands’), or papers published that makes you a leader in RNAi Therapeutics.

Oral presentation: Non-covalent peptide-based delivery systems (Divita, CRBM-CNRS-UMR5237, Montpellier, France)

This abstract concerning a non-covalent cell penetrating peptide-siRNA systemic delivery technology to me has firstly sentimental value. This is not a specific criticism of the work to be presented, a body of work that is buttressed by some credible data, but the abstract still reminds me of the early days when RNAi Therapeutics was hot…hot, hot, and all kinds of, sometimes wild, delivery claims were made: oral, blood-brain, all organs to name a few keywords.

While I wished that there was more excitement around RNAi Therapeutics right now as the negativity, particularly in the commercial arena, threatens to choke deserving technologies, the one benefit of RNAi being less hyped and exploited for fund-raising purposes by the biotech promotion machinery is that the overall scientific credibility index has increased. This can also be seen from the abstracts at this year’s OTS meeting.

Oral presentation: Investigating the potential of therapeutic oligonucleotides for pulmonary diseases (Clark, GSK)

GSK and AstraZeneca are probably the two Big Pharma companies most interested in RNAi/oligonucleotide Therapeutics for pulmonary diseases. This is an area with high unmet medical needs and new therapeutic approaches are needed here more than anywhere else. There are fundamentally two different approaches to knocking down genes in the respiratory tract: local delivery by aerosol inhalation, or through systemic delivery. Based on the abstract, GSK seems to be primarily interested in inhalation methods.

Among the companies having explored inhalation are Alnylam, ISIS’ respiratory disease spin-off Altair, and most recently Tekmira. It has become obvious that Alnylam’s naked siRNA approach (e.g. in ALN-RSV01) is sub-optimal and conjugation plus chemical modifications need to be applied to give such ‘semi-naked’ routes a chance. Altair meanwhile has closed down following phase II results with their naked MOE gapmer antisense candidate for asthma. Based on Tekmira’s track record of publicizing only meaningful scientific progress, Tekmira's aerosolized LNP approach is to be considered a serious contender for the leadership position in gene knockdown in the respiratory epithelium. Tekmira this year has presented data that its aerosolized LNPs retain the ability to knockdown genes in tissue culture. It is quite possible that GSK was/is the undisclosed Big Pharma collaborator for this program.

Silence Therapeutics, probably more by necessity than choice, takes a systemic approach towards gene knockdown in the lung using their intravenously administered lipoplexes (DACC). Actually, since Silence’s and Tekmira’s technologies may be best suited for endothelial and epithelial cell knockdown, respectively, the two approaches are complementary. It would make sense if AstraZeneca had some familiarity with Silence’s DACC technology.

Abstract #9: [3H]-radiolabeling of siRNA (Christensen, Novartis)

Abstract #86: Characterization of side reactions during the annealing of siRNA (Noll, Roche)

I list the two abstracts from Novartis and Roche here together because I believe they illustrate the cultural differences between Big Pharma and pure-play RNAi companies. While pure-play companies emphasize biology and developing new RNAi trigger and delivery solutions, the established pharmaceutical companies are apparently more concerned about manufacturing and pharmacology methods. It is obvious that manufacturing and pharmacology is an essential part of the game, and such work is also happening at pure-play companies and their outsourcing partners, but such work obviously does not address the rate-limiting challenges and Big Pharma, perhaps with the exception of Merck, willfully relies on accessing that from the pure-play companies.

Abstract #16: Inhibition of complement C6 synthesis in the liver using antisense oligonucleotides affects neuro-regeneration (Fluiter, Academic Medical Center, Amsterdam, Netherlands)

This abstract highlights that by knocking down a gene in the liver, one can have therapeutic benefits for a wide range of non-liver diseases, such as neurodegenerative diseases. This is not really surprising given that all organs almost exclusively depend on their development and function on what they are provided for by the blood. Proteins made in the liver constitute the majority of free proteins in the blood and consequently impact all organs. Complement proteins which play a critical in immunity are one example of such proteins. As most diseases contain a complement-related immune/inflammatory component, RNAi Therapeutics could be a tool for modulating a wide range of autoimmune and other hypersensitivity disorders.

This principle of inhibiting a target in one organ to address disease in others (see e.g. transthyretin amyloidosis) is in contrast to other, post-translational therapeutic drug modalities that target the liver for which the therapeutic benefit is almost always restricted to the liver. As such, the medical and commercial potential of RNAi delivery technologies that work well for gene knockdown in the liver is larger than widely appreciated.

Abstract #27: Thirteen week non-clinical testing of miravirsen in cynomolgous monkeys (Hildebrandt-Eriksen, Santaris)

This abstract concerns the toxicological evaluation of Santaris’ exciting phase II LNA anti-miR122, a LNA-modified phosphorothioate steric block antisense, for the treatment of HCV infection. Despite the successes of the recently approved protease inhibitors for genotype 1 HCV, there is still considerable unmet medical need, including for those with less drug-responsive genotypes or those high-risk patients that have failed on established therapies.

Presenting on home soil, the reported toxicities were in line with expected class effects of phophorothioate oligonucleotides, including slight, but relatively persistent clotting abnormalities which was not judged an adverse side effect because of the apparently small extent of the increase; reversible kidney toxicities at doses above 10mg/kg (the effective dose of miravirsen is likely between 2 and 5mg/kg); and finally some enlargements in macrophages which does not appear to be of too much concern. Note that because miravirsen is not intended for chronic use, this safety profile may be adequate. In addition to liver toxicity, it appears however that the kidney toxicity will be something to watch out for in the development of miravirsen.

The first phase II study of miravirsen has just completed enrolment according to clinicaltrials.gov and I look forward to learning about the results in due course.

Abstract #30: Lipid nanoparticle formulations of minimal-length shRNAs show potent inhibition of HCV-driven, liver-specific gene expression in mice (Johnston, Somagenics- in collaboration with Tekmira)

This abstract concerns the evaluation of 40-50 nucleotide hairpin RNAs with Tekmira’s LNP delivery technology for liver gene knockdown (in this case using HCV as a model system). It is not a surprise that the abstract shows that Tekmira’s LNP technology works with various RNAi triggers. The real new insight for Tekmira investors, however, is that Tekmira did not go into this litigation in a way that its access to payloads would be threatened as a loss of access to Alnylam’s RNAi triggers may very well be one of the outcomes that could facilitate a settlement. Instead, Tekmira must have been evaluating various RNAi trigger structures and presumably other nucleic acid payloads as well, and when it chose to exclusively license Halo-Bios multivalent RNAi triggers one has to assume that this was after an extensive evaluation of their safety and potency.

Whether there will be a similar arrangement with SomaGenics remains to be seen. Synthetic shRNAs are credible RNAi triggers and may in fact have some advantages over two-stranded approaches, e.g. highly efficient unimolecular annealing. However, their development has been held back by increased cost of goods associated with such long oligonucleotides and concerns about clogging up the RNAi enzyme Dicer (probably not an insurmountable challenge). A licensing decision may also depend on how broad SomaGenics' intellectual property is with regard to shRNAs. It is highly unlikely that SomaGenics has any gate-keeping claim in this area, and partnering with them would have to be driven by their shRNA-related know-how.

To be continued...(for part 2 click here)

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.

Breaking News: Pfizer Licenses DNA-directed RNAi Program for HCV from Tacere Therapeutics

Representing the first major DNA-directed RNAi licensing deal to date by Big Pharma, Tacere Therapeutics announced today licensing of its late preclinical-stage AAV-RNAi HCV program, TT-033, to Pfizer. Detailed terms were not disclosed, but Tacere will be eligible for up to $145 in development milestones, plus royalties on sales of drugs. Pfizer will assume all development work and cost.

TT-033 consists of an adeno-associated viral vector (AAV) expressing small hairpin RNAs (shRNAs) that are expressed from the viral vector. The shRNAs are processed by the RNAi machinery to yield small interfering RNAs that target the HCV RNA. This vector is devoid of any viral protein-encoding gene and has proven extremely efficient in transducing liver cells in mice and in many cases effecting almost complete gene knockdown. Tacere acquired the rights to TT-033 from Benitec when Benitec, due to funding problem, downsized and left the US in 2006. Benitec retains a stake in both Tacere and TT-033.

With today’s announcement, Pfizer is continuing is strategy of small, but deliberate RNAi technology deals. Tacere is a Bay Area company and close to Pfizer’s new Bioinnovation Center in South San Francisco. Last year, Pfizer made clear that it wants to become a major player in RNAi Therapeutics as part of its biotechnology initiative. It will now be interesting to watch whether Pfizer will take a broad technology license from Alnylam to protect all of its ongoing RNAi projects (Alnylam announced today that it is confident to close at least 2 major Roche-type technology licensing deals in the not-to-distant future).

The deal is a validation of AAV technology for gene therapy. AAV has shown great promise in addressing the RNAi delivery challenge for a number of organs, including the liver, brain, and eye. Benitec, Tacere, and Targeted Genetics currently have significant interests in AAV-mediated RNAi.

SomaGenics Reports on the Use of shRNAs for the Treatment of HCV

At the end of a day packed full with fun RNA science from the San Francisco Bay Area, Brian Johnston, President and CEO of SomaGenics presented some of his company’s data on the use of shRNAs for the treatment of HCV, historically one of the early therapeutic targets for RNAi.

Most shRNA approaches are based on DNA-directed expression of RNA hairpins in the nucleus that are then fed into the RNAi pathway. Somagenics’ approach is slightly different in that they are introducing the RNA directly into cells. Concentrating more on the science than IP issues here I only want to briefly remark that this probably overlaps quite a bit with the Hannon patents (licensed to both CytRx and Alnylam) on the use of shRNAs as RNAi triggers, both transcribed and synthetic.

The scope of the experiments were limited either to in vitro culture studies or its glorified in vivo counterpart, the hydrodynamic co-transfection experiment. Here, a large volume of RNA-containing liquid is injected into the tail vein of a mouse in a brief period of time, physically forcing the RNA into hepatocytes. It is of interest, that unlike the findings of the back-to-back papers of the Hannon and Rossi labs more than two years ago, hairpins (directed to the conserved HCV IRES) with minimal stems, 19 base-pairs, were generally more effective than those with extended 25 base-pair hairpins. While it has to be said that compared to the Hannon and Rossi papers which argued that the long stems that were processed by Dicer would increase RNAi efficacy, the sample size here was quite limited, it nevertheless shows that the processing of hairpins are often not as predictable as one would wish based on microRNA biology.

Johnston further reported that the shRNAs were at least as efficient as the corresponding siRNAs with some of the IC50s in the low picomolar range. I am curious how this will translate into in vivo uses for example because shRNAs, unlike classical siRNAs and Rossi-type two-stranded Dicer-substrates, contain the double-stranded RNA within one molecule which should make this structure thermodynamically quite stable. A potential disadvantage is that it is so much more expensive to synthetically make the ~42-55 nucleotide hairpin RNAs since cost and purity of manufacturing RNAs increases more than linearly after you reach a certain size, say 25 nucleotides. While these studies mostly used RNAs generated through biochemical synthesis in the test tube using recombinant phage RNA polymerases, for the clinic they are working together with Agilent to generate the “same” RNAs to scale. I say the “same” here since the phage polymerase leaves a triphosphate 5’ end while chemical synthesis does not. This is not a trivial issue here since 5’ modifications are known to influence RNAi processing. We will therefore have to wait how the transition from in vitro transcribed to synthetic shRNAs will affect the reproducibility of their data and potential concerns from the FDA. Similarly, some mention was made that shRNAs may tolerate RNA modifications less well than siRNAs, probably because they are subject to additional processing steps.

The use of more challenging animal models is also warranted before entering the clinic. It is therefore important for them to find suitable delivery solutions which they apparently have only started to. It was good to hear though hearing him mention Protiva/Tekmira’s SNALPs and Mirus Bio’s Dynamic PolyConjugates as probably the most advanced RNAi delivery platforms to the liver, reflecting my views on what’s out there in the literature. Overall, I like the fact that multiple RNAi approaches are in the pipeline to tackle HCV, namely siRNAs: Sirna/Merck; DNA-directed shRNAs: Tacere/Benitec and Nucleonics; and now synthetic shRNAs: SomaGenics. And maybe Tekmira/Protiva should harness the power of SNALP and officially nominate HCV as a clinical development program. I hope that the next 2 years should finally see programs moving into the clinic after HCV RNAi had been delayed in the wake of company-specific issues (Benitec; Merck/Sirna vs Protiva).

The Confusing World of AtuRNAi, Stealth siRNAs and mdRNAs (Part II)

(RNAi IP discussion continued from previous entry)

If long dsRNA had worked exactly in humans as it did in the worm and plants, then you would have expected an immediate flood of publications reporting the same. Long dsRNA for gene silencing, however, were impractical for most vertebrate cell applications due to the induction of non-specific cytokine responses that essentially shuts down most gene expression and therefore does not allow for targeted gene silencing. An exception may be embryonal cells which lack an interferon response and for which long dsRNA was reported to induce specific gene silencing first in zebrafish in 1999 (Wargelius et al. Biochem Biophys Res Commun. 263:156) and then in mice (a mammal) in late 2001 by the Filipowicz group (Basel, Switzerland).

These latter findings, however, were overshadowed earlier in 2001 by a publication from the Tuschl group in Germany, representing the culmination of a body of work he first started as a post-doc in the Bartel/Sharp labs during his time at the MIT, and then as an independent investigator at the Max-Planck Institute in Goettingen. While small RNAs were then known to derive from long dsRNAs, their molecular role in guiding the recognition and destruction of target mRNAs was only hypothesized and their structure mostly unknown. A breakthrough towards this understanding came by establishing a biochemical system in Drosophila (fly) lysates that recapitulated RNAi in the test tube (1999, MIT). One year later, they reported that during this reaction both the long dsRNA as well as the target mRNA is cut at 21-23 nucleotide intervals (MIT, 2000), thereby providing a link between dsRNA processing and mRNA targeting. In early 2001, then at the MPI, Elashir and colleagues in Tuschl’s lab further delineated the relationship between dsRNA processing and target mRNA cleavage in the Drosophila system, including the observation that the mRNA is cut around 10 nucleotides from the 5’ end of a 29 base-pair dsRNA (kind of Dicer-substrate). Importantly, by sequencing the 21-23 nucleotide RNAs by borrowing a cloning technique developed for the discovery of microRNAs around the same time, they found that the small RNAs were clustered consistent with long dsRNA processing into 21-23 base-pair DUPLEX RNAs. Moreover, the small RNAs were found to contain 5’ monophosphates and 3’ hydroxyl groups all consistent with the notion that long dsRNA was processed by an RNase III enzyme into 21-23 base-pair duplexes (reported to be the Dicer enzyme by Hannon in Cold Spring Harbor in the same month).

This led them to test whether small duplex RNAs were crucial functional intermediates between long dsRNA and mRNA cleavage, by synthesizing duplex RNAs and adding them to the Drosophila system. I quote: “Perhaps the 21-nt RNAs are present in double-stranded form in the endonuclease complex, but only one of the strands can be used for target RNA recognition and cleavage”. Indeed, this prediction turned out to be correct and synthetic duplex RNAs could silence mRNAs in this system, and duplexes with 2-3 nucleotide 3’ overhangs, the hallmark of the hypothesized RNase III-type processing, worked best. These data then formed the basis for the Tuschl I patent series to which Alnylam, RXi, and Sirna Therapeutics obtained co-exclusive licenses. My guess is that Alnylam actually would not mind if this patent wasn’t issued after all, since for some obscure reason UMass, unlike the Whitehead Institute, MIT, and MPI decided to grant RXi and Sirna co-exclusive licenses. Equally curious is the fact that while in the January 2001 paper the duplex RNAs were shown to work only in fly lysate, in the Tuschl I series, out of the blue, human cell studies are described. I could well imagine that the ultimately issued Tuschl I patent will be solely focused on the fly data, so that the first human siRNA description would be exclusive to the Tuschl II series (I would encourage you to read my 27 May, 2007 Blog “2007RNAi Therapeutics IP: The Importance of Being Tuschl” on this issue).

Clearly, work in human cells was ongoing at the time in Tuschl’s lab, and they conclude the fly paper in Genes and Development with the ominous statement: “The siRNAs may be effective in mammalian systems, where long dsRNAs can not be used because they activate the dsRNA-dependent protein kinase (PKR) response (Clemens 1997). As such, the siRNA duplexes may represent a new alternative to antisense or ribozyme therapeutics.” The compositions, methods, and uses of synthetic siRNAs in human cells are described in excruciating detail in the Tuschl II patent series, much of which has issued in the EU and US and is exclusively licensed to Alnylam.

A lot of the claims by other companies such as Silence Therapeutics and Invitrogen’s Stealth siRNAs center around the fact that Tuschl II emphasizes the 3’ overhangs of siRNAs, and that blunt-end siRNAs are therefore not subject to Alnylam’s IP estate, but completely ignore the fact that Tuschl, both in his fly and human work indeed tested blunt-end siRNAs, just that they did not perform as well as the overhang siRNAs. I speculate that the reason why Alnylam has not come out and spelt out this fact is because they may think that their equally exclusively licensed Kreutzer-Limmer patent series (use of short dsRNAs for gene silencing in mammals) provides even better coverage for the use of blunt-end siRNAs. Alnylam’s competitors, including Merck, have therefore focused their efforts of fighting Alnylam’s IP dominance on narrowing the scope of Kreutzer-Limmer, particularly in Europe, and have succeeded in doing so last summer to reduce the covered length to 15-21 nucleotides. However, a so called divisional patent application based on the Kreutzer-Limmer patent was granted in Europe in 2005 and has even broader claims than the original patent (15 to 49 base-pair duplexes). It is further ironical that a weakening of Kreutzer-Limmer would only strengthen Tuschl II’s scope. In addition, Tuschl II further covers modifications and conjugations to siRNAs, a claim which Alnylam has cemented by obtaining an exclusive license to the Crooke modification patent estate from ISIS.

While RXi may have marketed their recent StealthTM siRNA license from Invitrogen for therapeutic purposes, in my mind “StealthTM” siRNAs are nothing more than a marketing gimmick disguising the fact that these are 25 base-pair, blunt-end siRNAs with a supposedly magical pattern of base modifications. I would therefore not be surprised if Stealth failed to fulfill the non-obviousness criteria, in addition to the fact that I have not seen any evidence that Stealth, per se, performs any better, if not worse than the classical Tuschl siRNA design. What RXi probably won’t tell you is that Invitrogen has deemed it necessary to gain access to the Kreutzer-Limmer patents through a licensing agreement with Alnylam for the use of Invitrogen’s siRNAs for research applications only.

With regards to Dicer-substrate, licensed by both Nastech and Dicerna, I see practical value in that Dicer-substrates may be beneficial for RNAi delivery purposes in that they provide increased flexibility in covalently conjugating the Dicer-substrate to the delivery carrier, while siRNAs have to be reversibly conjugated, e.g. via disulfide linkages, to achieve the same. However, this does not guarantee the uniqueness of Dicer-substrate since a lot of the duplex length and the conjugation idea is subject to the pre-dating Kreutzer-Limmer and Tuschl patent series. Moreover, in his fly experiments with 29 base-pair duplexes, Tuschl already demonstrated “RNase III-substrate”. Hannon should also have relevance for Dicer substrate in that he was the first to describe Dicer to be the enzyme that mediates dsRNA processing in flies, and likely humans.

Hannon continued his work on the practical implication of dsRNA processing and was one of the first to explicitly describe the use of Dicer-substrates in humans in the form of DNA-directed hairpin expression cassettes driven by a Pol III promoter. While this 2002 paper in Genes and Development was as much inspired by the newly emerging knowledge on microRNA processing as much as by Tuschl’s 2001 findings, “Tuschl and colleagues first showed that short RNA duplexes, designed to mimic the products of the Dicer enzyme, could trigger RNA interference in vitro in Drosophila embryo extracts”), the European group (Brummelkamp et al.) that published in Science on the same subject the same month were mostly inspired by Tuschl: “We report here a new vector system, named pSUPER, which directs the synthesis of small interfering RNAs (siRNAs) in mammalian cells.” In any case, both Alnylam and RXi have gained access to the Hannon patents which touch on both DNA-directed RNAi and Dicer-substrate (synthetic or DNA-directed).

In a confusing turn of events, however, Hannon reported in 2005 that hairpins, in this case synthetic versions though, with longer duplex regions often worked better (= more potent and reliably) than the classical 19 base-pair hairpin design. This could be explained by the observation that the efficiency of RNAi should be enhanced by requiring a Dicer processing step since this is coupled to the RiSC-mediated gene silencing step. The fact that the original 19 base-pair hairpins, first thought to be processed by Dicer, were found to be inferior could be explained by their inefficient processing into siRNAs by some RNases not normally related to RNAi. Essentially the same conclusion was reached by a paper in the same issue of Nature Biotechnology from John Rossi’s group at the City of Hope, this time, however, by employing synthetic 25-30 base-pair duplexes (licensed to Nastech and Dicerna) instead of synthetic hairpins. For a discussion of Dicer-substrate science and IP, please refer to my October 31, 2007 Blog: “A new player in RNAi Therapeutics: Dicerna Pharmaceuticals”.

Besides RNA polymerase III-driven small hairpins, DNA-directed RNAi can also be initiated through more microRNA-like constructs. This was enabled by the elucidation of the microRNA silencing pathway and the trick basically is to design DNA vector constructs that will mimic one of the RNA intermediates during microRNA processing. These methods have the advantage that RNA polymerase II promoters can be employed with potential tissue-specific or other regulation. This should allow for potentially safer DNA-directed RNAi, although RNA polymerase III constructs have extreme knockdown potencies. Brian Cullen’s (Duke) and particularly Narry Kim’s (Seoul, Korea) groups have spear-headed these efforts, but I have not heard from companies yet specializing on the use of such RNAi constructs for therapeutic purposes. It is further likely that the original DNA-directed RNAi patents will be quite important for the commercialization of these later methods.

In addition to employing RNAi triggers that funnel into the RNAi pathway upstream of siRNAs, it is also theoretically possible to make use of at least two more intermediates functioning downstream of siRNA generation: single-stranded guide RNA that recognizes target mRNA within RiSC and a 3-stranded intermediate in which the passenger (=non-targeting) strand is interrupted based on findings from a number of groups, again almost simultaneously about 2 years ago, that the passenger strand was cut prior to guide RNA RiSC loading, analogously to how target mRNAs are cleaved.

The single-strand siRNA method is mostly investigated by ISIS for commercial purposes, probably because it feels that their IP position on single-stranded antisense RNAs would make them the dominant player in single-stranded RNAi. It should be kept in mind, however, that it was again the Tuschl group, known to be close to Alnylam, that first reported on single-stranded RNAi inducers (Martinez et al., 2002) and patents have been filed. Moreover, evidence so far suggests that single-stranded RNAs are only very inefficiently recognized by the endogenous RNAi machinery and it is doubtful that any potential advantages of single-stranded RNAs versus duplex RNAs would ever make up for the inferior potency. Patents covering 3-stranded siRNAs have been filed for by Nastech, although they have not been associated with any of the initial reports on siRNA passenger strand cleavage. It will therefore be interesting to determine the priority dates of the various discoveries, and probably more importantly, data as to the efficiency of these “meroduplex RNAs” (same initials as Nastech’s planned RNAi spin-out mdRNA) compared to other RNAi inducers. Such 3-stranded siRNAs may offer certain advantages with regard to conjugation chemistries and the fact that short RNA strands are cheaper to synthesize than larger ones, but until this is proven it will look just like another thinly disguised patent work-around attempt.

In summary, it is clear that it was Fire and Mello’s discovery on long dsRNAs as RNAi inducers in worms and Tuschl’s extensive body of work leading to the delineation of the classical duplex siRNA that opened up RNAi for therapeutic use. Many of the other developments are directly derived from both of these fundamental discoveries and require appropriate IP licenses. The combination of Tuschl II, Kreutzer-Limmer, and all the other patents it has either exclusive or non-exclusive access to, makes Alnylam the gate-keeper of RNAi Therapeutics. The exact terms of companies wishing to commercialize RNAi Therapeutics will vary depending on their co- or non-exclusive access to some of these fundamental patents, how far removed their exact siRNA derivatives are from the classical siRNA design as well as the ability to prove their utility. While this dominant IP position by Alnylam may make them unpopular and almost look like a bully, one should not forget that concentration and clarity of IP encourages investments particularly in the risky business of drug development and therefore will increase the likelihood of maximizing the therapeutic potential of the technology. It should also be said that Alnylam has been pretty good in de-risking RNAi technology, thereby benefitting the whole field of RNAi Therapeutics, in addition to granting access to RNAi technology through their licensing policy, although the terms will increase the longer you wait. Outside this core RNAi IP, other IP, particularly relating to delivery, but also access to validated targets will prove valuable, albeit much more fragmented.

In my last blog on this RNAi IP series, I would like to briefly discuss individual companies according to technology strength and IP position. Alnylam’s view on this issue will be presented in a special IP-focussed investor presentation on November 28 at the 19th Annual Piper Jaffray Health Care Conference and can be followed live or recorded by webcast on the company’s website.

Erratum: Please note that in my discussion of Dicer-substrate in the October 31, 2007 Blog: “A new player in RNAi Therapeutics: Dicerna Pharmaceuticals”, I mistakenly stated that Hannon’s long hairpin RNAs were DNA-directed, when they actually studied synthetic versions of these hairpins.

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.