Silenseed Lifts Secrecy of First Clinical RNAi Slow-Release Formulation

Last week saw the first peer-reviewed publication of the LODER RNAi delivery system developed by Israeli biotech Silenseed (Khvalevsky et al. 2013).  Following a ~17-subject phase 0/I study in pancreatic cancer patients that was initiated in 2010 and a more recent phase I study with siG12D in ~100 patients with locally advanced, unresectable pancreatic cancer, LODER (Locally Drug EluteR) had become the first matrix-assisted slow-release RNAi Therapeutic formulation in clinical development.  Nevertheless, I have struggled to understand the basics of the technology largely based on the patent literature which is often ambiguous.

Slow-release RNAi Therapeutics

Slow-release RNAi formulations promise to minimize the need for and frequency of repeat drug administrations.  This is of particular value when systemic delivery options are limited and direct access to the diseased site difficult and/or dangerous to the patient.  Ocular applications are one such area as large molecule drugs are commonly administered by intravitreal needle injections which carry a cumulative risk of injury to the retina and other complications when given every month or two as is common today.  Other areas are wound healing applications (e.g. sites of broken bones) where you basically get the chance to apply the RNAi just once and pharmacology is only needed for a few months, or for diseases of the pancreas where surgical manipulations carry the risk of potentially life-threatening pancreatitis.

While RNAi activity in non-dividing tissues is remarkably extended- it now seems that with stabilizing RNAi trigger modifications and efficient delivery you should be able to achieve potent silencing in the liver for 2 to 3 months following a single administration- slow-release strategies are attractive if the goal is to go beyond that.

 

Slow-release strategies typically involve matrices with embedded RNAi triggers that dissolve over time thus releasing the RNAi payload.  A major challenge is to find matrices and formulations that are not only biocompatible and bioresorbable, but that do so gradually.  This is because for most matrices you initially get a great burst of activity with a relatively quick drop-off in drug release thereafter.

LODER technology

At the basis of LODER technology is the PLGA (polylactic glycolic acid) workhorse of the medical device industry.  Staying with simplicity, it appears as if the RNAi triggers are simply embedded in the ~1-by-4mm pellets in unmodified Tuschl RNAi trigger format.  As the PLGA dissolves, the RNAi triggers are liberated with about 55% released in the first week, followed by another 25% or so over the next 60 days. 

The release kinetics appear acceptable and it is good to see the RNAi trigger being protected from degradation while inside the matrix (albeit not totally unexpected).  What seems to be far from optimal, however, is the apparent reliance on intracellular delivery by naked, unmodified RNAi triggers alone.  It seems that a combination of the PLGA matrix with self-delivering RNAi triggers or other cellular delivery formulations is called for (shameless self-advertisement : I can always be had for consulting projects).

siG12D LODER

Nevertheless, the PNAS publication by Silenseed reports good evidence of gene silencing in mouse models, one involving luciferase gene silencing in a transgenic mouse with luciferase expression in the liver, others involving ectopic and orthopic pancreatic tumor masses.

Importantly, performing insightful analyses of the relationship between distance from the implanted pellet(s) and gene silencing, it was found that gene silencing was marked in a sphere up to 2mm away from a pellet.  In the pancreatic cancer models, this was accompanied by local necrosis as expected from the specific knockdown of mutant KRAS which is thought to drive the majority of pancreatic cancers.  

Given that silencing is so locally restricted, this makes me wonder how best to apply the technology in the clinic.  In the pancreatic cancer trials by Silenseed, resectable and non-resectable cancer settings have been tested, in single- and multi-dose regimens.  Intuitively, I could imagine that post-operative settings are attractive, where the pellets are deposited at the border between cancer and normal tissue following the excision of the bulk tumor to kill any remaining cancer cells much in the same way that radiation therapy is often indicated following breast cancer surgery.  In cases of non-resectable pancreatic cancer, it would seem that stuffing the pancreas with LODER pellets once and then hope for the best (maybe in combination with gemcitabine and the like) is the go-for strategy.

I am encouraged by this new way of applying RNAi Therapeutics, but also see a number of simple ways of how to improve upon this first generation LODER formulation.  This would further resolve potential IP issues that come with the use of a prototypical Tuschl-type RNAi trigger. 

Impressions from the Abstracts of the 7th Annual Meeting of the Oligonucleotide Therapeutics Society (Part 2)

The OTS Meeting has just finished and it looks like it was a success in that meaningful progress, especially on delivery, but also specific development candidates was presented. As the press releases by Tekmira and Alnylam today showed, the abstracts do not necessarily reflect the full progress of the respective studies as these have been submitted months ahead of the conference and also because some groups may want to hold back with the most exciting data points.

For this reason, I will first summarize what I consider to be the highlights of today’s press releases, and then complete my review of the abstracts following a blog post from last week.

Tekmira and SomaGenics report ~300-fold knockdown of HCV in chimeric mouse model

In last week’s post I had mentioned that Tekmira and SomaGenics reported in their abstract #30 the successful use of 'short shRNAs' together with SNALP LNP delivery technology in a model of HCV. This model comprised of an HCV-luciferase reporter gene driven by a liver-specific promoter which is delivered on a plasmid before the application of the LNP-formulated shRNAs. A 90% knockdown at 2.5mg/kg was noted with these somewhat unorthodox RNAi triggers. This type of system is somewhat similar to knocking down a 'normal' gene in the liver, and as such the abstract did not suggest that studies were also performed in the context of real viral replication.

Today’s press release, however, revealed that the companies went an important step further, namely that they applied LNP-shRNAs in a chimeric mouse model for HCV infection. As a reminder, animal models of HCV infection are hard to come by. The best system may be the chimpanzee model, but it is obvious that this system does not lend itself to large numbers, is very costly, and it has been challenging also for ethical reasons to conduct these types of studies. More recently, chimeric mice have been developed in which the mouse liver is repopulated by transplanted human cells under selection pressure. Unlike the hepatocytes of mice and almost all other animals, these chimeric livers can sustain a form of HCV replication.

Having demonstrated 2.0-log and 2.5-log HCV viral knockdowns following one and two administrations of LNP-shRNAs, respectively, is therefore intriguing news. Similar to Santaris’ anti-miR122 antagonist, such a treatment should have significant potential to increase cure rates in treatment failure patients and those patients with genotypes not served well by current therapies.

Alnylam reports more potent LNPs and liver cell-targeted GalNac-siRNA conjugates

Alnylam issued an OTS-related press release today on three different lines of RNAi delivery research, two concerning LNPs, one to siRNA conjugates. As ionizable SNALP LNPs already incorporate a hepatocyte-targeting mechanism (ApoE), the interesting aspect of the GalNac-siRNA conjugates (Abstract #32) is that they may better lend themselves to subcutaneous administration compared to the mostly intravenously delivered LNPs- although this may be less so if the LNP delivery efficiencies are in the single to low double-digit micrograms per kg. The 5 mg per kg ED50 of GalNac-siRNAs puts them right around where some of today’s RNaseH antisense technologies are just in terms of amount of oligonucleotides administered.

Alnylam claims that these numbers make GalNac-siRNA conjugates serious candidates for clinical development. Still, because of the superior, 1000-fold increased efficacies of LNPs and the less frequent dosing that can be achieved with them, this comment may be Alnylam trying to portray themselves as having multiple realistic delivery options, when in fact they are relying for essentially all of their relevant pipeline candidates on Tekmira’s LNPs- my prediction is also that the 5th and final 5x15TM candidate will be SNALP delivered. This cautionary note may also apply to the reported 0.002mg/kg ED50 MD1 lipidoid formulation, developed in collaboration with Professor Dan Anderson from the MIT, although I have yet to see the lipid structures and formulations to really conclude this.

The news on the ‘3^rd generation reLNPs’, probably belonging to the SNALP line of research, similarly relates to further increases in LNP potencies. Here, a ~10-fold increase was reported in therapeutic window over ‘2^nd generation LNPs’, including those containing the contentious MC3 lipid, with an ED50 of below 0.005mg/kg and being well tolerated at the much higher 10mg/kg dosage. Whether Tekmira has a claim also on reLNPs remains to be seen.

Abstract #35: Preclinical development of sd-rxRNAs for fibrosis and retinal disorders (RXi Pharmaceuticals)

RXi Pharmaceuticals reports interesting progress with their self-delivering siRNA for dermal scarring with more than 2 weeks of CTGF gene silencing following single intradermal administration. Also based on Excaliard’s antisense work, CTGF seems to be a quite promising gene target for an orphan indication of solid market potential. RXi Pharmaceuticals would probably aim to improve upon Excaliard’s candidate by increasing the extent and duration of CTGF knockdown, making it potentially a once or twice drug administration approach instead of one involving multiple administrations every couple of days.

RXi expects to file the corresponding IND later this year.

Abstract #43: Characteristic aspects of skeletal muscle as a target organ for siRNA (Dainippon Sumitomo and Koken, both Japan)

I found this abstract particularly interesting because it indicates that Japanese pharma company Dainippon Sumitomo has a quite broad interest in RNAi Therapeutics. As a reminder, Dainippon Sumitomo is one of Silence Therapeutics’ larger RNAi target and delivery collaborators and we should hear about that particular relationship soon.

Koken meanwhile is a company which has an interest in providing delivery solutions for RNAi Therapeutics using its collagen-derived atelocollagen formulation.

Abstract #59: Dicer-substrate siRNA exhibit improved guide strand selection and stronger RISC Loading Complex formation compared to canonical siRNA (Rossi lab, City of Hope)

This study concerns the molecular comparison between Dicer-substrate RNAi triggers, as also practiced by Dicerna, and more conventional (Tuschl-type) RNAi triggers. The abstract suggests that Dicer substrates were more potent and more specific than the competition. A big sweeping comparison between these structures remains to be performed and published to put this issue to rest.

Abstract #69: Glucan particles for selective delivery of siRNA to phagocytic cells in mice (University of Massachusetts)

Glucan-encapsulated siRNA particles (GeRPs) made headlines and raised a few eyebrows a few years ago when a study published in Nature claimed these to be a viable oral delivery technology for targeting gene knockdown in phagocytic cells throughout the body. RXi was the licensee to that technology.

The present update concerns combining GeRP-like particles with the Endoporter peptide-based delivery system. The micrometer-sized GeRP particles are meant to be taken up by phagocytes with the amphiphilic Endporter chemistry supposed to overcome the ‘last mile’, that is RNAi trigger escape into the cytoplasm which has been a challenge especially with phagocytic cells. In principle, these ‘first-localize, then escape’ two-component formulations are reasonable to develop. Whether this particular one can solve the problem, again, remains to be seen. It is probably noteworthy that the present abstract does not mention the oral delivery of these particles.

Abstract #72: RNA-based pancreatic cancer therapy by local delivery of ant K-RASMT siRNA (Silenseed, Israel)

The Israeli company Silenseed has been quietly pursuing the development of its siG12D LODER slow-release siRNA matrix for the local treatment of pancreatic cancer (gene target: mutated KRAS). This candidate is unique in the clinical RNAi Therapeutics staple in that it uses a biodegradable polymer matrix to locally deliver siRNA not at once, but over a sustained period of about 8 weeks. The matrix is placed into the pancreatic tumor mass by ultrasound-guided procedure.

While the concept is sound in principle, it is still not clear whether Silenseed has considered all of its technical requirements such as the intracellular delivery of the RNAi trigger following its local release and distribution. It would not appear an insurmountable challenge, but I have seen groups pursuing promising concepts, but really addressing half the technical requirements only.

Abstract #97: miRNA turnover in Dicer knockout cells (Monash University, Australia and IDT)

This is an interesting abstract as it addresses the inherent stability of small silencing RNAs in mammalian cells. This stability is responsible for allowing us to often observe 4 weeks and more of RNAi gene silencing in non-dividing tissues after single RNAi trigger administration in vivo. With the more potent LNPs, every two months (intravenous) dosing is already within reach.

In this particular system, the authors removed the critical microRNA enzyme Dicer by genetic ablation in mouse embryonic fibroblast. With this, the observed decline in miRNA abundance reflects the decay of the largely RISC-incorporated single-stranded miRNAs following Dicer knockout. An average miRNA half-life of ~5 days was observed this way. It is likely that knocking out the microRNA pathway upstream of Dicer at the Drosha step would have yielded even slightly prolonged half-lives and this would have better reflected the fate of double-stranded RNAi triggers. It will be critical to find out by how much these half-lives can be further increased using siRNA chemistry, as Merck has done, and how much of it is determined by the protein turnover of the RISC complex which ultimately may be a rate-limiting step difficult to meddle with.