The Retina within Sight of RNAi Therapeutics

 From the highly prevalent diseases such as the age-related macular degenerations (dry and wet) to the rarer, but more numerous diseases such as the retinitis pigmentosa, the eye is the subject of a significant and growing unmet medical need.  From a genetic point of view, the application of RNAi Therapeutics to these diseases is very attractive.  However- yes, you guessed it- delivery challenges have made it difficult to exploit its potential.  In 2013, I have come across at least two pieces of evidence, one in synthetic and one related to DNA-directed RNAi, which make me believe that the time for ocular RNAi Therapeutics is nigh.

A bit of history

Following an early rush into ocular diseases that saw 3 wet AMD RNAi candidates, one each by Opko Health, Quark Pharmaceuticals (partnered with Pfizer), and Sirna Therapeutics/Merck (partnered with Allergan), speed into phase II and III studies, considerable doubts about their scientific foundations killed off the enthusiasm.  

Firstly, it was thought that the preclinical validations of the wet AMD candidates rested on TLR3-activation artifacts.  While I have not seen this claim more widely supported and chemical modifications and short double-strandedness should readily get around this issue, the more vexing question to me has been how were these either unmodified or simply 2’-O-methyl modified, unformulated RNAi triggers supposed to enter their target cells?

Moreover, for ddRNAi where Genable Technologies is readying an AAV-based candidate for retinitis pigmentosa to commence clinical trials, vector distribution following needle administration represents serious safety and efficacy issues.  Because the AAV and lentiviral gene therapy workhorses do not diffuse on their own from intravitreal injection sites to the back of the eye, ocular gene therapy has largely involved subretinal injections which a) is a relatively dangerous procedure that can seriously harm retinal architecture and integrity, and b) limits vector diffusion and therefore therapeutic activity to a small area surrounding the subretinal administration site.

Smartly evolved AAV can penetrate from the vitreous into the retina

In order to develop viral vectors capable of overcoming the physical barriers between the vitreous and retina, Dalkara and colleaguespublished earlier this year a study on the selection of AAV2 variants capable of doing just that. 

The selection process of this now popular method of directing AAV to various tissues and cell types starts out with large libraries of more or less randomly mutated AAV variants.  In each selection round, the viral DNA is extracted from the target cells and re-amplified for the next round of selection such that the AAV variants that most efficiently transduce the target cells eventually become highly enriched.

 

The 7m8 AAV that they identified was thus capable of broadly transducing retinal cells following intravitreal administration in mice, from the inner ganglion cells to the outer retinal pigment epithelial cells.  In non-human primates, 7m8 also greatly enhanced retinal penetration from the vitreous compared to old gold standard AAV serotypes.  However, retinal cells were not transduced as broadly as in mice suggesting that either additional candidates from mouse evolution experiments need to be characterized in non-human primates or that the selection itself should be performed in non-human primates.

I view such progress exceptionally promising for retinal degenerative diseases in which rescue of only a fraction of cells should be therapeutic (e.g. by getting rid of a mutant mRNA that leads to the death of the cell expressing it), but potentially also for the more challenging disease settings where RNAi action would probably have to occur in the majority of cells in order to be therapeutic.

 

Along these lines, after deciding on the best AAV variant, an important task will be to determine the more precise percentage of cells of each cell type that can be transduced to then match them up with potential indications.

Sticky self-delivering RNAi triggers penetrate deeply into the retina

In the field of synthetic RNAi Therapeutics, a peer-reviewed paper came out by RXi Pharmaceuticals (Byrne et al.) which confirmed earlier claims that self-delivering RNAi triggers (sd-rxRNAs as they call their own versions) were able to fully penetrate the retinal cell layers in both the mouse and rabbit following intravitreal injections.  The rabbit is an important model because their eye volume, believe it or not, is close to ours (~1.5ml vs ~5.0ml).  Formal proof of RNAi-mediated gene silencing was then confirmed in the mouse.

The self-delivering RNAi triggers had guide strands of ~19 nucleotides and passenger strands of less than 15 nucleotides.  An extended phosphorothioate (PS) single-stranded tail on the guide and additional hydrophobic modifications such as sterol groups were supposed to enhance tissue penetration and functional cell uptake.  The 6-8nt PS tail obviously has been borrowed from standard antisense chemistry.  Therefore, a battery of tests was performed to exclude visual abnormalities resulting from these extensive modifications.  No notable tox findings were made.

Despite the similarities in chemistry, sd-rxRNAs appear to be superior to ISIS-type PS-ASOs (RNaseH mechanism).  This is because in the Byrne et al. study, ~3 microgram RNAi trigger was required for 50% gene knockdown, whereas 50 micrograms phosphorotioate antisense oligos were required for a 50% knockdown of MALAT as presented by ISIS at OTS 2013 in Naples (report still available).  To wit­, MALAT as a nuclear RNA can be expected to be far more susceptible to RNaseH ASOs than your average mRNA, so the potency difference could be even bigger.  Also, it would be interesting to determine the safety implications of administering 3 micrograms versus 50 micrograms or whatever the equivalent dosages in humans.

The potency difference would have been much bigger still if RXi were not so married to their less than 15bp double-strandedness, a misguided decision driven by old IP considerations (when the Kreutzer-Limmers were still a concern).  I therefore expect other RNAi companies with more active R&D to thank RXi for the validation and come up with more optimal RNAi reagents for ocular applications.

Ocular RNAi Therapeutics- keep an eye on it in 2014 and beyond.

ISIS Demonstrates Wider Utility of RNaseH ASOs for Nuclear Targets

While research astounds us on a daily basis with unexpected discoveries, sometimes it is what we don’t know and haven’t bothered to ask is what is astounding.  One example of the latter in the field of Oligonucleotide Therapeutics is the poor understanding of which tissues and cell types and therefore disease indications are most appropriate for a particular (delivery) approach based on the ability to engage targets there.   

Antisense Therapeutics, over 30 years in the making, has been the biggest violator of this principle.  Smug in the belief that delivery is not necessary, the approach has been to just apply the oligonucleotide and then pray that it will go to the right place and work its magic, especially in cancers.  Only after decades, the field through much clinical trial and error has come to the realization that the liver and kidney may be pharmacologically favored target organs.

In a long overdue tour-de-force, Hung and colleagues from ISIS Pharmaceuticals recently published in the journal Nucleic Acid Therapeutics a detailed investigation of the global biodistribution and RNaseH knockdown efficacy of phosphorotioate antisense chemistries (PS-ASO) following systemic application: Characterization of target mRNA reduction through in situ RNA hybridization in multiple organ systems following systemic antisense treatment in animals. This parallels a similar study for the direct application of this chemistry to the CNS presented at this year's OTS meeting which has yielded the surprising insight of the broad CNS distribution of PS-ASOs following focal administration. 

As a result of the latest research, a roadmap of target organs was created.  Importantly, through the application of newer RNA immunohistochemistry methods rather than the old harvesting and mashing up organs, the study looked at the specific cell types within an organ that were amenable to RNaseH knockdown.  This is important in at least two ways.  Firstly, it allows us to reject a potential target in organs where bulk knockdowns have shown a rather deep knockdown, but where the detailed organ analysis shows that the particular cell type in which one desired the knockdown does not show such a knockdown (e.g. kidney).  Secondly, it allows one to reconsider targets and cell types within organs for which bulk knockdowns have not been observed (e.g. the small intestines).

Another valuable piece of insight of the study was that it compared the old, second-generation 2’ MOE chemistry with the higher affinity locked nucleic acid chemistry version pioneered by Santaris (in this case the cET ISIS knock-off version of LNAs).  In addition to increasing the knockdown potency in traditional tissues such as liver, kidney, and adipose tissue, the chemistry allows for appreciable knockdowns in some less traditional tissues such as muscles.  Unfortunately, the direct comparison between 2’ MOE and LNAs was only performed in mice and at the very high 50mg/kg dose.  In the non-human primate study, also at a very high (35mg/kg) dose, no such direct comparison was  performed and from this, it seems that the new organs enabled by the higher-affinity chemistries were limited to the muscle and lung.

Why Marina Biotech could be the 2014 high-flyer

Regular readers will notice that I have shifted some of my investment attention to Marina Biotech.  The main reason for this is that this company which is considered by many to be dead, actually owns the rights to a high-affinity ASO chemistry (CRN) of a potency that is equivalent to Santaris’ LNAs and probably superior to ISIS’ cET while the market cap of Marina is just one-thousandth that of ISIS Pharmaceuticals.  Even when one considers that the in vivo safety (especially) and potency evaluations lag behind the others due to the budget constraints of Marina Bio, I believe it is a risk worth taking given the enormous valuation gap and the fact that CRN PS-ASO biodistributions and activities can be assumed to be similar to the competing chemistries.

What is more, Marina Bio is pursuing a program in type I myotonic dystrophy which represents the sweet spot of indications uniquely facilitated by these chemistries: muscle as a new druggable target organ and still shielded from superior RNAi competition; a rare, severe orphan disease; and a toxic nuclear RNA.

Largely depending on the recapitalization strategy (partnering first before capital raise or vice versa), this program together with SMARTICLE RNAi delivery and access to usiRNAi triggers, has made me accumulate 1.5% of the company with the intention of increasing my position.  Of course, financial success can only happen if other investors share my view that we should therefore give Marina Bio another chance.  As always, invest at your own risk and according to your unique financial circumstances.

A shameful title

If you re-read the title of the paper and even the entire publication, you may be forgiven for going away with the impression that it is open season for RNaseH knockdown in muscles and other tissues and organs.  This is far from the truth as the ‘exemplary’ target chosen in the study was the nuclear non-coding RNA MALAT.   This is because a high-profile Nature publication by ISIS Pharmaceuticals itself (Wheeler et al. 2012) has shown that whereas largely cytoplasmic m-e-s-s-e-n-g-e-r RNAs (i.e. RNAs encoding for proteins as even a decent high-school kid will know) expressed in muscles were entirely recalcitrant to RNase H knockdown, the mutated nuclear retained DMPK underlying myotonic dystrophy was susceptible to such action.  Curiously, while Wheeler et al. was cited in the Hung paper, the authors failed to point out this important and very obvious caveat.

This can be no innocuous omission as ISIS Pharmaceuticals in one of their patent applications has expressed the striking difference between mRNA and nuclear RNA druggability by PS-ASOs as follows (highlights are mine):

Reduction of Nuclear-Retained RNA

Data provided herein demonstrates that sensitivity to cleavage by ASOs is dramatically increased for a nuclear-retained RNA making it possible to reduce nuclear-retained targets in tissue that has low uptake of oligonucleotide by a pharmacologically relevant amount. For example, out of the more than 4,000 transcripts that Isis has targeted by antisense, MALAT1, a non-coding, nuclear-retained RNA, is demonstrated to be one of the most sensitive targets for antisense oligonucleotide/RNase H inhibition. The data demonstrate a great number of oligonucleotides targeting over the majority of the transcript that inhibit by more than 50% in vitro. The data also demonstrates very low IC50 values in multiple cell types. Half-life studies have also shown that the MALAT1 is stable over a period of at least 10 hours. Subcutaneous administration of oligonucleotide targeting MALAT1 at doses commensurate with other oligonucleotide drugs (e.g., liver targeting drugs) achieved pharmacologically relevant reduction of MALAT1 in skeletal and cardiac muscle. Dosing at 50 mg/kg biweekly for 3.5 weeks achieved a 89% and 85% reduction in gastrocnemius and quadriceps, respectively, and 54% reduction in heart (as compared to 95% reduction in liver). Pharmacologically relevant reduction of MALAT1 has also been achieved in tumor xenograft models.

As a member of the Oligonucleotide Therapeutics Society, it greatly saddens me that the related journal is letting ISIS Pharmaceuticals get away with the highly misleading, and simply wrong title.  There is no arguing around it.  Followers of the competitive oligonucleotide therapeutics investment arena know that the game here is to make RNaseH antisense appear much more widely applicable than it actually is.  What is more, it was at the 2011 OTS meeting in Boston where the ISIS CEO Stan Crooke in his keynote made the ignonimous statement that ‘mipomersen has no side effects’.  

I strongly suggest to the society and the journal Nucleic Acid Therapeutics which are supposed to foster the development of the technology broadly to keep a watchful eye on the growing corporate influence, especially by 'generous sponsors' ISIS and Alnylam Pharmaceuticals.   

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. 

Delivery Advance Illustrates Influence of Cosmetics Skin RNAi Therapeutics

The skin has always been a target organ of considerable interest to the RNAi Therapeutics industry due to its apparent accessibility for delivery purposes plus the fact that there are various unmet needs ranging from the severe genetic disease (e.g. epidermolysis bullosa, pachyonycia congenita) to cosmetic desires.  Interestingly, it is the latter that in many ways is driving skin RNAi Therapeutics these days.

Motorized microneedle array with unprecedented silencing efficacy

    

In an important advance in the rate-limiting area of delivery, Hickerson and colleagues from TransDerm and various other collaborators recently published 80% gene silencing efficacy in a mouse model for epidermal gene expression using a motorized microneedle array borrowed from the cosmetics industry (in particular the Triple-Mby BomtechElectronics of cosmetics hot-spot South Korea).  This compares to 50% and 33% gene silencing in the same model using simple (static) microneedle arrays and intradermal needle injection, respectively, before.  Accordingly, this represents a 2.5 to 3.5-fold increase in gene silencing efficacy when considering how much of the undesired target protein you are left with!

I have to admit that I did not double-check that indeed the same siRNA sequences and self-delivering RNAi trigger modifications were used in the various studies which could have affected results.  However, since these results have all been reported by TransDerm and the goal of TransDerm was to compare delivery efficacies of various technologies, I am willing to accept the comparability claim by the authors. _­

The trick with the motorized microneedle array appears to be that following penetration of the stratum corneum barrier motion (oscillation) allows for a larger volume of drug to be deposited in the epidermis than with a static needle array.  Moreover, the depth of administration can be adjusted for optimal epidermal delivery and to make it pain free as well, unlike the original high-pressure hypodermic needle attempts by TransDerm.   With this, it should be possible to deposit low single-digit milligram of RNAi triggers to an area the size of a tip of a thumb- which is quite a bit.

A possible limitation of such microneedle arrays is that the administration itself causes microinjuries to the skin.  Therefore, you want to make sure that you do not end up making things worse, especially in applications where wound healing and restoration are the goal.  Since the technology is apparently used in the beauty industry already, it is unlikely that its application will leave insightly scars and the likes.

I look forward to seeing a technology like motorized microneedle arrays in conjunction with self-delivering RNAi trigger formats being used in the clinic.  Initially, the technology is most amenable to applications where the focus is on locally defined areas such a skin parts prone to blistering.  However, taking advantage of imaging technologies and 3-D printing, I envision a future in which the technology would also be possible to treat large areas of the skin, if not the entire body surface.  As TransDerm illustrates, combining the capabilities of existing technologies from disparate areas often enables the biggest advances.

RXI-109 for dermal anti-scarring now available under the ‘Specials’ provision in the EU

Anybody that has gone to a dermatologist knows how blurred the lines between medical and cosmetic applications have become when it comes to the skin (cosmeceutical concept).  Taking advantage of the regulatory grey zone, it is skin applications that are leading the charge in the commercialization of RNAi gene silencing in WoMan.  Following a claimed treatment for skin blemishes marketed as Britena Whitening & Anti-blotch Cream by Biomics (partnered with Benitec on HepB), it is now RXi Pharmaceuticals that has signed a distribution agreement for its dermal anti-scarring drug candidate RXI-109 with Ethicor. 

The goal of this arrangement is to drive early sales based on an exception of European drug legislation that allows for the use of experimental drugs prior to proper marketing authorization.  All it apparently takes is a judgment call by the treating physician.  I can see the point of this ‘Specials’ provision for severe, orphan diseases as a form of compassionate use when there is intriguing early clinical evidence of efficacy and safety, but for an anti-scarring treatment, mmh...you can easily see how consumers willing to take risks in the quest for beauty will make their physician give them an injection of the stuff.

But then again, when you see how much unproven, potentially harmful potions and lotions are being sold on the cosmetics market, it is hard to argue why you should make an exception with RNAi as long as care is being taken that somewhat riskier (depending on chemistry) systemic exposures remain low and yours truly does not have to pay for it via increased insurance premiums.  I guess my biggest problem with all this is that the company distributing RXI-109 calls itself ‘Ethicor’ just as I get nervous when somebody starts a sentence with ‘to be honest’ and what follows is more often than not a lie.