Marina Biotech Finds Success with Local Bladder Cancer Approach

Marina Biotech this week reported pre-clinical data supporting the use of local, intravesicular siRNA delivery for the treatment of nonmuscle invasive bladder cancer. The publication in Molecular Therapy comes weeks after this early-stage program was partnered with Swiss pharmaceutical Debiopharm which will be responsible for funding the rest of the development.

According to the National Cancer Institute, there are about 70,000 new cases of bladder cancer in the US alone, most of them related to smoking. Because bladder cancer typically starts at the epithelium lining exposed to the urine, simple surgical resection is often sufficient to stop the disease in its tracks. Unfortunately, in some cases the cancer recurs and progresses into a more aggressive, muscle-invasive form of cancer contributing to the 14,700 deaths from bladder cancer annually (US number). Current strategies to prevent recurrence include the instillation of immuno- and chemotherapeutics into the bladder. When this fails, the bladder often has to be removed. Clearly, more treatment options are needed to limit the progression of bladder cancer into the more aggressive forms and maybe also to provide an alternative for bladder removal once more aggressive cancers have developed.

In the present study, Seth and colleagues first screened a range of RNAi triggers against the cancer-related genes polo-like kinase 1 (PLK1) and survivin and were able to demonstrate that Marina’s usiRNA chemistry platform allows for very satisfactory knockdown efficacies with IC50 values in the 10 to 30pM range (bladder cancer cell lines in tissue culture). This triggered widespread apoptosis and, in the case of PLK1, a strong reduction in cell viability. The data therefore add to the notion that PLK1, incidentally also the target in Tekmira’s phase I clinical program TKM-PLK1, may be the single-most promising RNAi cancer target, a view apparently shared by Marina scientists as evidenced in the Discussion part of the paper. Nevertheless, survivin may still be a highly desirable drug target for sensitizing cancer to chemo- and radiation therapies.

The selected siRNAs were then formulated into positively charged multilamellar liposomes of around 100nm. Due to the negatively charged proteoglycan-rich urothelium, a formulation with a lipid containing a guanidinium group was deemed particularly promising in being able to penetrate the 6-7 cell-layered urothelium. Accordingly, these formulations, when instilled into the bladder, were able to very efficiently suppress the growth of nonmuscle invasive bladder cancers in mouse models of the disease. Although equally highly efficient in vivo knockdowns were reported, 90-95% with 1mg/kg (relative to body weight), in my mind there are still some unanswered questions as to how much of that merely reflected the reduction in the size of the tumor.

In terms of safety, no untoward reactions to the formulations were reported. One important reason for this should be the lack of systemic exposure from such treatment. The authors also stated that they were not able to find evidence of innate immune stimulation despite careful investigation. Ideally this analysis would have included a positive control, but then again, also taking into account the 5’ RACE analysis for sequence-specific target cleavage, the overall evidence strongly supports that RNAi knockdown was a main contributor to the in vivo efficacy. Actually, in the case of bladder cancer, having a bit of local immune stimulation may very well be a good thing as I was surprised to learn that bathing the bladder with a solution containing the Calmette-Guerin bacterium (BCG), the famous vaccine against tuberculosis, is part of the current standard of care for superficial bladder cancer and is thought to work by stimulating the immune system. Who knows, Marina may even want to adapt BCG to trans-kingdom RNAi for bladder cancer to combine the RNAi and BCG mechanisms of action into a single agent.

Study Adds yet Another Indication Suitable for Current RNAi technologies

Marina Biotech has made it into an art to select development programs amenable to their current technologies. Given the challenges of systemic RNAi delivery, in a relatively short period of time, the company has already identified and progressed three cancer applications where local approaches can probably be used: familial adenomatous polyposis (FAP; ingested trans-kingdom RNAi), bladder cancer (intravesicular LNP), and most recently malignant ascites (intraperitoneal LNP). Together with developments elsewhere in the field, this clearly shows that the time for RNAi Therapeutics is Now: a range of solid cancers (e.g. liver, lung, FAP, bladder, melanoma), hypercholesterolemia, hemorrhagic fever viruses (Ebola, Marburg), TTR amyloidosis, PHN, AMD, Huntington’s disease etc etc.

Aptamer-siRNAs: Another Shot at RNAi Therapeutics Delivery

There has been a trickle of papers lately describing the use of aptamers for the functional delivery of siRNAs such as for cancer and HIV. Aptamers are highly folded, 35-100 nucleotide long RNAs that can bind protein targets with relatively high affinities and specificities. One way of thinking about them is as the RNA equivalent of antibodies. Aptamers already are being tested as a therapeutic class of its own where they are typically designed to neutralize extracellular targets, with already one aptamer (Macugen) approved for wet AMD.

As such, aptamers should lend themselves for targeting associated therapeutic siRNAs to cells of interest, in a sense functioning like antibodies and small molecules that have likewise been recruited for targeted RNAi delivery. What distinguishes an aptamer-siRNA combination, however, is the promise of having to simply use only RNA synthesis to generate a pharmacologically viable siRNA therapeutic, obviating the need for complicated formulation technologies. Furthermore, when it comes to repeat-administration such a system may cause inherently little adaptive immunogenicity.

The reason why I have been somewhat skeptical on this technology is that like with so many siRNA targeting approaches, getting to the cell of interest is just a first step, and it is not obvious to me how after e.g. receptor-mediated endocytosis the rather large aptamer-siRNA conjugate would be able to cross the negatively charged lipid bilayer to get into the cytoplasm for incorporation into the RNAi-induced silencing complex (RiSC).

Nevertheless, a recent study in Nature Biotechnology (Dassie and colleagues: “Systemic administration of optimized aptamer-siRNA chimeras promotes regression of PSMA-expressing tumors”) suggests that competitively low mg/kg dosages of intraperitoneally injected prostate-specific membrane antigen- (PSMA) targeted aptamer-siRNAs can efficiently knock down the popular cancer target PLK1 in a mouse xenograft model of prostate cancer. The study is a follow-up of a 2006 paper published in Nature Biotech by the same group from the University of Iowa (McNamara and colleagues: “Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras”) where intratumorally injected- i.e. not systemically administered- PSMA-targeted aptamer-siRNAs showed very efficient inhibition of tumor growth in the same model system.

Since systemic administration is deemed to be necessary for an siRNA therapeutic against prostate cancer, the investigators reasoned that they could achieve such delivery by increasing the potency of the aptamer-siRNA by primarily improving siRNA potency through siRNA design (changing an initially blunt siRNA into a Tuschl-type 3’ overhang type) and then attaching the ubiquitous PEG to increase circulation times so that the aptamer-siRNA would have an increased chance of finding its target. As hoped for, both strategies substantially improved in vivo performance. Impressively, PEG addition increased the half-life of the molecule from less than 35 minutes to over 30 hours (!) and this was accompanied by improved silencing and tumor inhibition. The 3’ overhang siRNA (actually it was a Dicer substrate- more on this later) was also much better than the original blunt-ended version. While most aptamer-siRNAs are bi-molecular which reduces the maximum length of RNA to be synthesized, a unimolecular precursor microRNA mimic performed best. This could due to increased stability of an intramolecular duplex and/or a more “natural” appearance to the RNAi machinery. Practically, however, bimolecular conjugates may be preferable as RNA synthesis becomes exponentially less efficient with size and is also for this reason that the authors further reduced the length of the aptamer from the earlier study.

Overall, all of the many controls that they were probably asked for by the reviewers confirmed the specificity of the results: the therapeutic effect correlated very well with the degree of knockdown, both in vitro and in vivo; binding and silencing was only observed in PSMA-bearing cells; no innate immunostimulation that might explain the anti-cancer effect was detected; 5’ RACE showed that there was in vivo RNAi activity. Finally, only ~21nt siRNAs were detected following administration of the Dicer-substrate RNAi triggers which suggests highly efficient Dicer processing. Generally, it has to be said that while the shorter, traditional siRNAs have many advantages in terms of specificity and immunity, Dicer-substrates may be ideally suited for conjugate approaches such as this, as Dicer-processing would liberate and thereby activate the functional siRNA whereas Argonaute loading, in theory, should be diminished by a direct conjugate to the siRNA (however, strategies such as reversible disulfide bonds might work for such a configuration).

As an aside, the studies are further validation of PLK1 as a very good target for RNAi Therapeutics in oncology. PLK1 is one of the most highly over-expressed genes in cancer, and knockdown studies have shown that cancer cells are very sensitive to the reduction in PLK1 levels while normal/healthy cells, even if transfected with PLK1 siRNA are unaffected. PLK1 is also the target for a SNALP cancer therapeutic candidate developed by Tekmira for solid cancers that is slated for IND next year (Alnylam with a 50:50 opt-in right until start of phase II).

In a sign that there is also commercial interest in aptamer-siRNAs, the leading aptamer company Archemix and Dicer-substrate company Dicerna recently agreed to collaborate on aptamer-siRNA delivery. Archemix, which shares a building with Alnylam, similarly chose to collaborate with heart- and muscle-focussed miRagen on the delivery of microRNA therapeutics. Archemix’ sudden move into small RNA therapeutics is also quite interesting given their failed IPO attempt and speculations of a reverse takeover of Silence Therapeutics.

So where do I think aptamer-siRNA delivery technology stands? I’m still somewhat skeptical and would like to see more of these studies from various laboratories. An important question that was posed by an accompanying News and Views article from Alnylam scientists (which btw makes it very likely that the paper was reviewed by them) is whether the surprising cytosolic uptake of the RNA is a peculiarity of the PSMA antigen or could be a more widely mechanism for presumably endosomal escape that could be exploited. Studies into the precise molecular mechanism of the uptake, as with all RNAi delivery systems, are needed. One could also imagine that to enhance uptake, membrane-active agents may be added to the PEG-aptamer-siRNA, although this would be contrary to the initial concept of a simple design. In summary, the more varied approaches being explored, the better for RNAi Therapeutics. For now, aptamer-siRNAs are just one of those to be watched.